Chapter 4. Normal Physiology of the Hypothalmic-Pituitary-Thyroidal System and Relation to the Neural System and Other Endocrine Gland
The activity of the thyroid gland is predominantly regulated by the concentration of the pituitary glycoprotein hormone, thyroid stimulating hormone (TSH). In the absence of pituitary or of thyrotroph function hypothyroidism ensues. Thus, regulation of thyroid function in normal individuals is to a large extent determined by the factors which regulate the synthesis and secretion of TSH. Those factors are reviewed in this chapter and consist principally of thyrotropin releasing hormone (TRH) and the feedback effects of circulating thyroid hormones at the hypothalamic and pituitary levels. The consequence of the dynamic interplay of these two dominant influences on TSH secretion, the positive effect of TRH on the one hand and the negative effects of thyroid hormones on the other, result in a remarkably stable concentration of TSH in the circulation and consequently little alteration in the level of circulating thyroid hormones from day to day and year to year. This regulation is so carefully maintained that an abnormal serum TSH in most patients indicates the presence of a disorder of thyroid gland function. The utility of TSH measurements has been recognized and its use has remarkably increased in recent years due to the development of immunometric methodologies for the accurate quantitation of this protein in serum. Thus, an understanding of the regulatory influences on TSH secretion underlies both normal thyroid physiology and the pathophysiology of thyroid diseases.
This chapter is organized into two general sections. The first portion reviews basic studies of TSH synthesis, post-translational modification and release. The second deals with physiological studies in humans which serve as the background to the diagnostic use of TSH measurements and reviews the results of TSH assays in a pathophysiological context.
TSH is a heterodimer consisting of α and β subunits tightly, but non-covalently, bound. [1, 2] While the molecular weight of the deduced amino acid sequence of mature α plus TSH β subunits is approximately 28,000 Da, additional carbohydrate (15% by weight) results in a significantly higher molecular weight estimate based on sizing by polyacrylamide gel electrophoresis. The α subunit is common to TSH, follicle stimulating hormone (FSH), luteinizing hormone (LH), and chorionic gonadotropin (CG). The β subunit confers specificity to the molecule since it interacts with the thyroid cell TSH receptor and is rate-limiting in the formation of the mature heterodimeric protein. However, the free β subunit is inactive and requires noncovalent combination with the α subunit to express hormonal bioactivity. The linear sequence of human α subunit is represented by 92 amino acids including 10 half-cystine residues, all of which in disulfide linkage. The human TSH β (hTSH β) subunit contains 118 amino acids, as predicted by complementary DNA sequences, but hTSH β isolated from pituitary gland has an apoprotein core of 112 amino acids, due to carboxyl-terminal truncation during purification.
The production rate (PR) of human TSH is normally between 50 and 200 mU/day and increases markedly (up to >4000 mU/day) in primary hypothyroidism; the metabolic clearance rate (MCR) of the hormone is about 25 ml/min/m2 in euthyroidism, while is significantly higher in hyperthyroidism and lower in hypothyroidism. [3] The PR of free α subunit is about 100 μμg/day, increases only about twice in primary hypothyroidism and/or in post-menopausal women and decreases (about to one half) in hyperthyroidism. [4] The PR of free TSH β subunit is too low to be calculated in all hyperthyroid and in most euthyroid subjects, while is 25-30 μg/day in primary hypothyroidism. [4] The MCR of free subunits is 2-3 times faster than that of TSH, being about 68 ml/min/m2 for α and 48 ml/min/m2 for β subunit. [4]
The human α subunit gene is located on chromosome 6 and the TSH β gene on chromosome 1. [5] The structure of α subunit gene has been determined in several animal species. [6, 7] The genes of each species are approximately of the same size and similarly organized in four exons and three introns. The human gene is 9.4 kilobases (kb) in lenght, with three introns measuring 6.4 kb, 1.7 kb and 0.4 kb, respectively. The TSH β subunit gene has been isolated in mouse[6], rat[8], and humans. [9, 10] At difference with the α subunit, the organization of the TSH β gene is somewhat variable between the different species. The rat and the human genes are organized in three exons, while the mouse gene contains two additional 5'-untranslated exons. The first exon is untranslated, the leader peptide and the first 34 aminoacids are encoded by the second exon, while the third exon represents the remaining coding region and 3'-untranslated sequences. A single transcription start has been identified in hTSH β gene, while the rat and the mouse genes contain two starting sites separated by approximately 40 base pairs (bp); most transcription begins from the downstream site, which corresponds to the location of the human transcriptional start. A schematic representation of TSH β gene is reported in Fig. 4-1.
Figure 1. Thyrotropin β (TSH β) gene structure and mutations found in patients with congenital central hypothyroidism (modified from McDermott et al. [35])
![Thyrotropin β (TSH β) gene structure and mutations found in patients with congenital central hypothyroidism (modified from McDermott et al. [35])](./figures4/figure1.gif)
The pre-translational regulation of TSH synthesis and secretion is a complex process, detailed in the next paragraphs. The formation of mature TSH involves several post-translational steps including the excision of signal peptides from both subunits and co-translational glycosylation with high mannose oligosaccharides. [11, 12] As the glycoproteins are successively transferred from the rough endoplasmic reticulum to the golgi apparatus, the trimming of mannose and further addition of fucose, galactose and sialic acid occurs. [13] The α subunit has two and TSH β one asparagine (N)-linked oligosaccharides showing typical biantennary structure fully sulfated in bovine and half-sulfated in human TSH. [2] The primary intracellular role of these glycosylation events may be to allow proper folding of the α and TSH β subunits permitting their heterodimerization and also preventing intracellular degradation. [13, 14] On the basis of crystallographic studies on hCG and other glycoprotein hormones, an homology model of the tridimensional structure of TSH has been proposed. [15] This model (Fig. 4-2) predicts for both α and β subunits the presence of two β-hairpin loops (L1 and L3) on one side of a central "cystine knot" formed by three disulfide bonds, and a long loop (L2) on the other side. Interestingly, the presence of a central cystine knot is observed not only in glycoprotein hormones, but is characteristic of an expanding superfamily of growth factors, including transforming growth factor-β (TGF-β), nerve growth factors (NGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF) and others. [2]
Figure 2. Schematic drawing of human TSH, based on a molecular homology model built on the template of a hCG model[14]. The α-subunit is shown as checkered, and the β-subunit as a solid line. The two hairpin loops in each subunit are marked L1, L3; each subunit has also a long loop (L2), which extends from the opposite site of the central cystine knot. The functionally important α-subunit domains are boxed. Important domains of the β-subunit are marked directly within the line drawing (crossed line, beaded line and dashed line): For further details the reader is referred to Grossman et al.[2]. (Reproduced from Grossman at al. [2], with permission).
![Schematic drawing of human TSH, based on a molecular homology model built on the template of a hCG model[14]. The α-subunit is shown as checkered, and the β-subunit as a solid line. The two hairpin loops in each subunit are marked L1, L3; each subunit has also a long loop (L2), which extends from the opposite site of the central cystine knot. The functionally important α-subunit domains are boxed. Important domains of the β-subunit are marked directly within the line drawing (crossed line, beaded line and dashed line): For further details the reader is referred to Grossman et al.[2]. (Reproduced from Grossman at al. [2], with permission).](./figures4/figure2.jpg)
Proper TSH glycosilation is also necessary to attain normal bioactivity, [16] a process which requires the interaction of the neuropeptide thyrotropin releasing hormone (TRH, Fig. 4-3), with its receptor on the thyrotroph. [17-19] The requirement for TRH in this process is illustrated by the fact that in patients with central hypothyroidism due to hypothalamic-pituitary dysfunction, normal or even slightly elevated levels of radioimmunoassayable, but biologically subpotent TSH are found in the circulation in the presence of a reduced free T4. [20, 21] Chronic TRH administration to such patients normalized the glycosylation process enhancing both its TSH-receptor binding affinity as well as its capacity to activate adenyl cyclase. This, in turn, can normalize thyroid function in such patients. [22] On the other hand, enhanced TSH bioactivity is invariably found in sera from patients with thyroid hormone resistence. [23] Moreover, variations of TSH bioactivity (mostly related to different TSH glycosylation) have been observed in normal subjects during the nocturnal TSH surge, in normal fetuses during the last trimester of pregnancy, in primary hypothyroidism, in patients with TSH-secreting pituitary adenoma and in non-thyroidal illnesses. [23, 24] Glycosylation of the molecule can also influence the rapidity of clearance of TSH from the circulation. Taken together, these findings have lead to a new concept of a qualitative regulation of TSH secretion, mainly achieved through both the transcriptional and posttranscriptional mechanisms involved in TSH glycosilation. [25]
Specific amino acid sequences in the common α and TSH β subunits are critical for the heterodimerization, secretion and bioactivity of mature TSH. These sequences include highly conserved segments which are essential for TSH receptor binding and biological activity (see Ref 2 for an extensive review). The peptide sequence 27CAGYC31(cysteine-alanine-glycine-tyrosine-cysteine) is highly conserved in TSH β, LH β, hCG β and FSH β and is thought to be important in subunit combination. [26] In Japanese families with an autosomal recessive form of hypothyroidism due to TSH deficiency, a point mutation in exon 2 has been identified which changes the glycine residue in the 27CAGYC31sequence to arginine. [26, 27] If a combination of α and mutant TSH β subunit mRNAs are injected into Xenopus oocytes, no intact TSH heterodimer is formed, unlike what occurs when a similar experiment is performed with a normal TSH β mRNA. This illustrates an important role of the CAGYC peptide in influencing the tertiary structure of the TSH β subunit, thereby facilitating the heterodimerization process. All known inherited TSH β gene mutations responsible for familial isolated central hypothyroidism are listed in Table 4-1 and depicted in Fig.4.1. The most frequent mutation is a homozigous single base deletion in codon 105 (C105D, 114X) leading to unstable heterodimer. [28-36] It has been suggested that the prevalence of this mutation in the general population could be higher than previously believe. [35]
Table 1. Mutations of the TSHβ gene responsible of isolated central hypothyroidism: effects on TSH heterodimer formation.
|
Mutation of TSH β gene |
Consequence of mutation on TSH heterodimer formation |
|---|---|
|
G29R [26, 27] |
Prevents dimer formation modifying the CAGYC region |
|
E12X [28] |
Truncated TSH β subunit unable to associate with α chain |
|
C105Δ, 114X [29, 31, 32] |
Change of amino acid sequence in the “seat belt” region leading to unstable heterodimer |
|
Q49X [32, 33] |
Truncated TSH β subunit forming a bio-inactive heterodimer with the α chain |
|
IVS2+5→A[34] |
Base substitution at intron 2 (position +5) with shift of the translational start point to an out of frame position of exon 3 resulting in a truncated transcript |
|
C85R[37] |
T to C transition at codon 85 of exon 3, resulting in a change of cysteine to arginine, preventing the formation of a functional heterodimer with the α-subunit |
The understanding of the relationship between molecular structure and biological activity of TSH recently allowed the synthesis of TSH variants designed by site-directed mutagenesis that block in vitro and in vivo TSH activity. It is conceivable that these new antagonists of TSH may offer novel therapeutic alternatives in treating hyperthyroidism. [37]
Recently, a new thyrotropic hormone represented by a heterodimer of two new glycoprotein subunits (A2/B5) has been identified in human pituitary and called thyrostimulin. [38] The precise physiological role of thyrostimulin remains to be identified, although it has been hypothesized that it could account for the residual stimulation of thyroid gland observed in patients with central hypothyroid. [39]
The major regulators of TSH production are represented by the inhibitory effects of thyroid hormone[40] and by the stimulatory action of TRH. As shown in Fig. 4-4, T3 acts via binding to the thyrotroph nuclear T3 receptor, and T4 mainly acts via its intra-pituitary or intra-hypothalamic conversion to T3, although a direct negative effect of T4 independent from local T3 generation has been recently reported on TSH β gene expression. [41] Both regulate the synthesis and release of TSH at the pituitary level, as well as indirectly affecting TSH synthesis via their effects on the synthesis of TRH and other neuropeptides. TRH is the major positive regulator of hTSH β gene expression and mainly acts by activating the phospatidylinositol-protein kinase C pathway. Other hormones/factors are also implicated in the complex regulation of TSH-β gene expression, as detailed below.
Figure 4. Basic elements in the regulation of thyroid function. TRH is a necessary tonic stimulus to TSH synthesis and release. TRH synthesis is regulated directly by thyroid hormones. T4 is the predominant secretory product of the thyroid gland, with peripheral deiodination of T4 to T3 in the liver and kidney supplying roughly 80% of the circulating T3. Both circulating T3 and T4 directly inhibit TSH synthesis and release independently; T4 via its rapid conversion to T3. SRIH = somatostatin.
In animal models, thyroid hormone administration is followed by a marked decrease of both α and TSH β subunit mRNA, [42, 43] but TSH β is suppressed more rapidly and more completely than α subunit. In humans with primary hypothyroidism a paradoxical increase of serum TSH concentration has been observed shortly after beginning thyroid hormone replacement therapy, followed later by TSH suppression. [44] The precise mechanism for this phenomenon has not been fully elucidated: it could be due to a generalized defect in protein synthesis as a consequence of hypothyroidism, or to the presence of a still unrecognized stimulatory thyroid hormone cis-acting element (see below). Thyroid hormone regulation of TSH β subunit transcription is complex and, at least in the rat and mouse, involves control of gene transcription at both start sites of the gene [44-51] (Fig. 4-5). Studies of the human, rat and mouse TSH β genes have demonstrated that they contain DNA hexamer half sites with strong similarity to the T3 response elements (TREs) found in genes which are positively regulated by thyroid hormone [52-54] (see Chapter 3). The sequences in the TSH β gene are diagrammed in Fig. 4-5 and their similarity to the typical hexamer binding sites in positively regulated genes and in the rat α subunit gene is seen by comparison to the TRE sequences from positively regulated genes[55] (Fig. 3-14, Chapter 3). In keeping with this concept, T3 exerts similar negative activity on rat GH3 cells transfected with plasmids constructs containing the putative negative TRE of rat TSH β or containing a half-site motif of the consensus positive TRE. [45, 48, 55-57] The molecular biology of negative regulation by thyroid hormone is currently under active investigation. The conserved TRE-like sequences are the best candidate site on the TSH gene to which the T3 receptor (TR) binds. The subsequent binding of T3 to TR-DNA complexes suppresses transcription of both α and TSH β subunit genes. [45, 48, 55, 58] The inhibitory effect of thyroid hormone is observed with all α and β isoforms of TR, but TR-β2 (a TR isoform with central nervous system-restricted expression) has the greatest effect. [59] This in vitro observation is in keeping with a series of in vivo data obtained in transgenic and knockout mice with generalized or pituitary-selective expression of mutated TR isoform genes. Knockout mice for TR-α1 develop only minor abnormalities in circulating T4 and TSH concentration[60], while mice lacking both β1 and β2 isoforms (β-null) develop increased serum T4 and TSH level, but retain partial TSH suppression by T3 administration. [61, 62] Mice selectively lacking TR-β2 isoform develop hormonal abnormalities similar to TR-β-null animals, indicating a key role of TR-β2 as mediator of T3-dependent negative regulation. [63]On the other hand, the residual T3-dependent TSH suppression observed in mice lacking TR-β isoforms suggests that TR-α1 may partially substitute for TR-β in mediating T3 suppression: accordingly, mice lacking all (α and β) TR isoforms develop dramatic increase in circulating T4 and TSH concentration, indicating that a complete expression of all TR isoforms are required for normal regulation of the hypothalamic-pituitary thyroid axis. [64-66] A detailed analysis of the effect on pituitary-thyroid function of different transgenic mice harboring different double homozygous or combined heterozyous deletions of both the TR-α and TR-β genes has been carried out by Weiss et al. [67] The results obtained provided clear evidence for: 1) unliganded TR-α or TR-β are not absolutely necessary for the upregulation of TSH in hypothyroid animals (see also below for a further discussion of this point); 2) TR-β but not TR-α is sufficient of thyroid hormone mediated down-regulation of TSH; 3) TR-α may partially substitute for TR-β in mediating a partial thyroid hormone dependent TSH suppression. In the attempt to dissect the molecular mechanisms underlying the T3-dependent inhibition at the pituitary (TSH) and/or hypothalamic (TRH) level, experimental models of mice with pituitary selective expression of mutant TR have been developed. [66, 67] Indirect evidence supporting a key role of TR in TSH regulation is also provided by the demonstration of a somatic mutation of TR-β in a TSH-secreting pituitary adenoma, [68] which could be responsible for the defect in negative regulation of TSH by thyroid hormones typical of these tumors. TR-β integrity is also necessary for the T3 modulation of hypothalamic TRH expression. [69]
Figure 5. DNA sequences of the putative TREs in the rat, mouse, and human TSH β subunit gene promoters. A comparison of the proximal promoter regions of the rat, mouse, and human TSH β subunit genes is shown. The straight arrows denote TRE consensus half-sites identified by functional and TR binding assays. The first exons (relative to the downstream promoter for the rat and mouse genes) are shaded, and the bent arrows denote the sites of transcription initiation. Note a nine-nucleotide deletion in the human gene relative to the rodent genes indicated by the triangle just 5' of the transcriptional start site. (Reproduced from Chin et al, [45] with permission.)
![DNA sequences of the putative TREs in the rat, mouse, and human TSH β subunit gene promoters. A comparison of the proximal promoter regions of the rat, mouse, and human TSH β subunit genes is shown. The straight arrows denote TRE consensus half-sites identified by functional and TR binding assays. The first exons (relative to the downstream promoter for the rat and mouse genes) are shaded, and the bent arrows denote the sites of transcription initiation. Note a nine-nucleotide deletion in the human gene relative to the rodent genes indicated by the triangle just 5' of the transcriptional start site. (Reproduced from Chin et al, [45] with permission.)](./figures4/figure5.jpg)
The negative transcription conferred by TSH β TRE sequences is retained even if they are transferred to a different gene or placed in a different position within a heterologous gene. [55, 70-72] This suggests that the negative transcriptional response to thyroid hormone is intrinsic to this TRE structure. In contrast with positive TREs, little is known about the mechanism of T3-dependent negative regulation of genes like TSH β. Data discussed above clearly show the crucial role of the TR-β in the negative regulation of TSH synthesis. Like positive TREs, it has been recently established that TR binding to DNA is required for negative gene regulation. [73] Early experiments suggested that unliganded TR homodimers stimulate the expression of TSH β, [45] (a behavior appearing a mirror image of the silencing effect on positive TREs), but the methodology employed was not adequate to study the low level of basal TSH β transcriptional activity. More recently, the use of CV1 cell lines containing the TSH β-CAT (chloramphenicol acetyltransferase) reporter allowed a more accurate study of the molecular mechanisms involved in the liganded TR suppression. [74] In this experimental system TSH β gene suppression was dependent on the amounts of T3 and TR, but unliganded TR did not stimulate TSH β activity, suggesting that TR itself is not an activator. Moreover recruiting of co-activators and co-repressors were shown to be not necessarily essential, but required for full suppression of TSH β gene. [74]
In contrast to the potentiating activity exerted on stimulatory TREs, retinoid X receptors (RXR) either unliganded or in combination with retinoic acid (RA) block thyroid hormone-mediated inhibition of TSH β gene, possibly through competition with the TR-T3 complex binding to DNA. [56, 74-76] However, RA is also able to suppress TSH β gene production when bound to RAR and RXR interacting with response elements separate from negative TREs. [77, 78] Taken together, these findings imply that distinct mechanisms are involved in thyroid hormone dependent inhibition and stimulation. Indirect support to this concept derives from the identification in a patient with selective pituitary thyroid hormone risistence a TR mutation associated with normal or enhanced function on stimulatory TRE in peripheral tissues, but defective function on inhibitory TREs of TSH β and TRH genes. [79]
An other peculiar feature of the negative TSH β TRE is that its 5' portion (Fig 4-4 and 4-8) displays high homology with the consensus sequence of binding sites for c-Jun and c-Fos, which heterodimerize to form the transcription factor called AP-1. This makes the negative TSH β TRE a "composite element" able to bind both thyroid hormone receptors and AP-1. [72, 79-81] Since AP-1 antagonizes in vitro the inhibition exerted by thyroid hormone, it may act in vivo as a modulator of TRH-dependent regulation of TSH β gene. [82] The role of other important TSH β gene activity modulators (such as Pit-1 and its splicing variants) will be discussed later. Other abnormalities of the mechanisms involved in the negative feed-back on TSH by thyroid hormones could be involved in rare pathological conditions of difficult identification and diagnosis. One of these conditions could be the case reported of an adolescent with congenital hypothyroidism escaped to the screening detection for inappropriately normal serum TSH concentration, in whom evidence of hypersensitive pituitary-thyroid feed-back axis was provided. [83]
Since unliganded TR does not behave as an activator of TSH β gene, other mechanisms are involved in the increase of TSH production observed in hypothyroidism. In the hypothyroid rat the TSH production is increased 15 to 20 fold over that in the euthyroid state. This can be attributed to the stimulatory effects of TRH (see below) unopposed by the negative effects of T3; moreover, besides the transcription rate per cell, there is a 3 to 4 fold increase in the absolute number of thyrotrophs in the hypothyroid pituitary. [84] Electron microscopic studies have shown near total depletion of secretory granules in the thyrotrophs of hypothyroid animals, a change that is reversed soon after administration of thyroid hormone. [85]
The acute administration of T3 to the hypothyroid rat causes a rapid and marked decrease in the level of serum TSH. [40, 86](Fig. 4-6) This decrease occurs prior to the decrease in pituitary α and TSH β mRNAs. [43, 86-88]. During the period that circulating TSH is falling, pituitary TSH content remains unchanged or increases slightly. [89] The suppression of TSH release is rapid, beginning within 15 minutes of intravenous T3 injection, but is preceded by the appearance of T3 in pituitary nuclei. [89] In the experimental setting in the rat, as the bolus of injected T3 is cleared and the plasma T3 level falls, nuclear T3 decreases followed shortly by a rapid increase in plasma TSH. Both the chronological and quantitative relationships between receptor bound T3 and TSH release are preserved over this time. [89]
Figure 6. Time course of specific pituitary nuclear T3 binding and changes in plasma TSH in hypothyroid rats after a single intravenous injection of 70 ng T3 per 100 g of body weight. Since the maximal capacity of thyroid hormone binding in pituitary nuclear proteins is about 1 ng T3/mg DNA, the peak nuclear T3 content of 0.44 ng T3/mg corresponds to 44% saturation. The plasma level falls to about 55% of its initial basal level by 90 minutes after T3 injection demonstrating that there is both a chronological and a quantitative correlation between nuclear T3 receptor saturation and suppression of TSH release. (From Silva and Larsen, [374] with permission)
![Time course of specific pituitary nuclear T3 binding and changes in plasma TSH in hypothyroid rats after a single intravenous injection of 70 ng T3 per 100 g of body weight. Since the maximal capacity of thyroid hormone binding in pituitary nuclear proteins is about 1 ng T3/mg DNA, the peak nuclear T3 content of 0.44 ng T3/mg corresponds to 44% saturation. The plasma level falls to about 55% of its initial basal level by 90 minutes after T3 injection demonstrating that there is both a chronological and a quantitative correlation between nuclear T3 receptor saturation and suppression of TSH release. (From Silva and Larsen, [374] with permission)](./figures4/figure6.jpg)
The mechanism for this effect of T3 is unknown. As discussed before, Suppression of basal TSH release is difficult to study in vitro. Accordingly, the T3 induced blockade of TRH-induced TSH release has been used as a model for this event. This T3 effect is inhibited by blockers of either protein or mRNA synthesis. [90, 91] The effect is not specific for TRH since T3 will also block calcium ionophore, phorbol ester or potassium-induced TSH release. [92, 93] Furthermore, T3 will also block the TRH-induced increase in intracellular calcium which precedes TSH release. [94] Thus, T3 inhibits TSH secretion regardless of what agent is used to initiate that process.
T4 can cause an equally rapid suppression of TSH via its intrapituitary conversion to T3. [86] (Fig. 4-4) This T4 to T3 conversion process is catalyzed by the Type 2 deiodinase. (see Chapter 3). An effect of T4 per se can be demonstrated if its conversion to T3 is blocked by a general deiodinase inhibitor such as iopanoic acid. [86, 95] In this case, the T4 in the cell rises to concentrations sufficient to occupy a significant number of receptor sites even though its intrinsic binding affinity for the receptor is only 1/10 that of T3. A similar effect can be achieved by rapid displacement of T4 from its binding proteins by flavonoids. [96] It seems likely, however, that under physiological circumstances the feedback effects of T4 on TSH secretion and synthesis can be accounted for by its intracellular conversion to T3.
The effect of suppressive doses of T3, T4 and triiodoacetic acid on serum TSH has been recently re-evaluated by ultrasensitive TSH assays. [97] TSH suppression was shown to be a complex, biphasic, nonlinear process, with three temporally distinct phases: phase 1, a rapid TSH suppression, starting after 1 h and lasting for 10-20 h; phase 2, slower suppression, starting between 10 and 20 h and lasting for 6-8 weeks; and phase 3, with stable low TSH level (<0.01 mU/L). This pattern of thyroid hormone suppression of TSH is reproducible and independent of the basal thyroid status or the thyroid hormone analog used.
Based on the analyses of the sources of nuclear T3 in the rat pituitary (Fig 3-11, Chapter 3), one would predict that approximately half of the feedback suppression of TSH release in the euthyroid state can be attributed to the T3 derived directly from plasma; the remainder accounted for by the nuclear receptor bound T3 derived from intrapituitary T4 to T3 conversion. [86] Various physiological studies in both rats and humans confirm this concept in that a decrease in either T4 or T3 leads to an increase in TSH. The effect of T4 is best illustrated in the iodine deficient rat model (Fig. 4-7). In this paradigm, rats are placed on a low iodine diet and serum T3, T4, and TSH quantitated at frequent times thereafter. [98] Despite the fact that serum T3 concentrations remain constant, there is a marked increase in TSH as the serum T4 falls. In humans, severe iodine deficiency produces similar effects. [99] The most familiar example of the independent role of circulating T4 in suppression of TSH is found in patients in the early phases of primary hypothyroidism in whom serum T4 is slightly reduced, serum T3 is normal or even high normal range, but serum TSH is elevated [100, 101] (Table 4-2).
Figure 7. Serum T3, T4, and TSH concentrations (mean ± SD) in rats receiving a low iodine diet (LID), with or without potassium iodide (KI) supplementation in the drinking water. (From Riesco et al, [98] with permission)
![Serum T3, T4, and TSH concentrations (mean ± SD) in rats receiving a low iodine diet (LID), with or without potassium iodide (KI) supplementation in the drinking water. (From Riesco et al, [98] with permission)](./figures4/figure7.jpg)
Table 2. Mutations of the TSHβ gene responsible of isolated central hypothyroidism: effects on TSH heterodimer formation.
|
TSH (mU/L) |
||||
|---|---|---|---|---|
|
T4 |
T3 |
Basal |
After 200 μg TRH |
|
|
Groupa |
(μg/dl) |
(ng/dl) |
||
|
Results are mean ± SD. a Patients were categorized according to the severity of thyroid disease based on serum T4 concentrations. (Adapted from Bigos et al, [101] with permission) |
||||
|
Control |
7.1±0.9 |
115±31 |
1.3±0.5 |
11±4.6 |
|
1 |
6-9 |
119±40 |
5.3±2.3 |
39±15 |
|
2 |
4-6 |
103±20 |
13±10 |
92±50 |
|
3 |
2-4 |
101±35 |
63±56 |
196±120 |
|
4 |
<2 |
43±28 |
149±144 |
343±326 |
TRH is critical for the synthesis and secretion of TSH either in the presence or absence of thyroid hormones. Destruction of the parvo-cellular region of the rat hypothalamus, which synthesizes the TRH relevant for TSH regulation, causes hypothyroidism. [102, 103] Hypothalamic TRH synthesis is in turn regulated by thyroid hormones and thus TRH synthesis and release is an integral part of the feedback loop regulating thyroid status (see Fig. 4-4). TRH also interacts with thyroid hormone at the thyrotroph raising the set-point for thyroid hormone inhibition of TSH release. [102] The data supporting these general concepts are reviewed in subsequent sections.
TRH is synthesized as a large pre-pro-TRH protein in the hypothalamus and in several tissues, such as the brain, the β cells of the pancreas, the C cells of the thyroid gland, the myocardium, reproductive organs including the prostate and testis, in the spinal cord and in anterior pituitary. [102, 104-109] Recent investigations employing sophisticated techniques such as fast atom bombardment mass spectrometry and gas phase sequence analysis showed that most TRH immunoreactivity found in extrahypothalamic tissues is actually accounted by TRH-immunoreactive peptides displaying different substitutions of the aminoacid histidine of authentic TRH. [108] These TRH-like peptides could be active in autocrine/paracrine networks involving also extrapituitary TSH secretion, [108] but a detailed discussion of this topic is beyond the purposes of this chapter, which is intended to focus on the regulation of pituitary TSH production. This is dependent only on TRH synthesized in the paraventricular nuclei (PVN), although paracrine and autocrine activity has been recently described for TRH secreted in the anterior pituitary. [110] The human pre-pro-TRH molecule is a protein of 29 kDa containing 5 progenitor sequences for TRH. [111-113] These five peptides consist of a gln-his-pro-gly peptide preceded and followed by lys-arg or arg-arg di-peptides. The basic di-peptides are the cleavage sites for release of the tetra-peptide progenitor sequence. The glycine residue is the source of the terminal amide for the proline residue of TRH (Fig. 4-3). In addition to the pro-TRH peptides which are released from the pre-pro TRH molecule, intervening non-TRH peptides which have potential physiological function are co-released. [114] In particular, the prepro-TRH fragment 160-169, also known as hST10, TRH-enhancing peptide and Ps4 [115, 116] is able to stimulate TSH β gene expression and to enehance the TRH-induced release of TSH and prolactin (PRL) from the pituitary [115-118]. Ps4 and Ps4 high affinity receptors have been recently shown within several extrapituitary neural tissues and other endocrine systems (mainly in pancreas and in male reproductive system), and targeted pre-pro TRH gene distruption results in hyperglycemia besides the expected hypothyroidism. [115] An other pre-proTRH peptide (fragment 178-199) inhibits in vivo and in vitro basal and corticotropin-releasing hormone (CRH)-stimulated corticotropin (ACTH) release, and increases the in vitro inhibition of ACTH release by dexametasone. [119, 120] These data suggest that preproTRH 178-199 may be a modulator of ACTH secretion, although the physiological relevance of this phenomenon remains to be clarified. The prepro-TRH processing is mostly mediated by the prohormone convertases PC1 and PC2, and takes place during axonal transport after removal of the signal peptide. [116] Subsequent cleavages occur as the peptides move down the axon toward the nerve terminal, from which TRH is released into the hypothalamic-pituitary portal plexus. [102, 103] The TRH-producing neuron bodies are densely innervated by both catecholamine and neuropeptide Y (NPY) containing axons which may also regulate the synthesis of the pre-pro-TRH molecule (see below, “TSH in pathophysiological states – Nutrition” for more details on NPY and the hypothalamic-pituitary-thyroid axis). Somatostatin containing axons also contribute to the negative regulation of TRH synthesis. [121]
Thyroid hormones exert strong negative regulation of TRH synthesis at hypothalamic level. [122-126] This regulation is observed in vivo exclusively in the parvo-cellular division of the PVN[122, 124] (whose neurones contain functional TR isoforms α1, β2 and β1[127]), while in tissues outside the central nervous system expressing the TRH gene, negative regulation by thyroid hormone is absent. [128] TR β2 is the key isoform responsible for T3-mediated feedback regulation by hypophysiotropic TRH neurons[129]. Increases in TRH mRNA levels occur during primary or secondary hypothyroidism and decreases in TRH mRNA result from implantation of a small crystal of T3 adjacent to the PVN. [124] The physiological source of the T3 causing down regulation of TRH mRNA in the hypothalamus is the subject of ongoing investigations. Somewhat surprisingly, the PVN does not contain the Type 2 5' iodothyronine deiodinase which is thought to be the source of at least 80% of the intracellular T3 in the central nervous system [86, 130] (see Chapter 3). However, studies with T3 containing mini-pumps implanted into thyroidectomized rats indicate that, for normalization of circulating TSH and hypothalamic pre-pro-TRH mRNA, T3 concentrations about twice normal have to be maintained in rat plasma. [125] Thus, for both systems (TRH and TSH), feedback regulation requires a source of T3 in addition to that provided by the ambient levels of this hormone. While this T3 seems likely to be produced locally from T4, the anatomical location of such a process has not been identified. It is possible that T4 to T3 conversion occurs elsewhere in the CNS and the T3 is transported in synaptosomes to the PVN. [131]
The synthesis of TRH is under complex transcriptional control sharing several similar mechanisms (such as negative regulation by thyroid hormone) with the TSH β gene. The human TRH gene is located on chromosome 3 (3q13.3àq21) [132]; the 5' flanking sequence of the TRH gene has potential glucocorticoid and cyclic AMP response elements (GRE and CRE). [113] There are also potential negative TREs located in this portion of the gene which offer regulatory sites for thyroid hormone control of TRH gene transcription. The thyroid hormone negative regulatory elements of TRH gene are localized in its 5' flanking element (-242 to +54 bp). Four sequences within this region exhibit high degree of homology with consensus sequences for TRE half-sites (AGGTCA) and two of them show also homology with elements implicated in negative regulation by thyroid hormone of TSH β gene. [128] In the absence of thyroid hormone, unliganded TR exert a paradoxical stimulation of the TRH promoter, [132, 133]and other factors appear to concur with thyroid hormone in TRH gene inhibition. [134] Co-transfection studies with mutated TRs with distinct abnormalities of T3 binding associated to in vivo thyroid hormone resistance provided evidence for lowered inhibitory action on TRH gene expression by mutant TR-T3 complexes, as well as for their dominant negative effects on WT TR-T3 inhibition. It has been therefore suggested that these mechanisms likely contribute to the elevated serum TSH observed in the syndrome of thyroid hormone resistance. [135] Furthermore, a sequence downstream the transcription start site of murine TRH gene is essential in the negative regulation, although it does not contain any thyroid hormone binding site: [136] this suggests the cooperation of TR with unknown factor(s) interacting with DNA sequences downstream the transcription start. The negative effects of TR-T3 complex on TRH gene expression appear to be counterbalanced by stimulatory effects of AP-1 proteins (c-Fos/c-Jun), probably through protein-protein interactions between TR and c-Fos and/or c-Jun altering the binding capacity of TR and preventing the inhibitory action of TR-3 complex. [128]
The TRH gene promoter contains potential binding sites for cAMP response element (CRE) binding protein (CREB), as assessed by homology with standard CRE consensus sequence, and both human and rat TRH genes are positively regulated by cAMP. [128] One of the potential CREs of TRH promoter is a sequence that has overlapping TRE/CRE bases –53 to –60 bp (TGACCTCA) (Fig. 4-8) [128]. Recent studies provided evidence for competitive interactions of TRβ1 molecule and CREB at the overlapping TRE/CRE in the TRH promoter. [128] Constructs of TRH promoter with mutations in this overlapping site prevented both the inhibition by TR-T3 complex and the paradoxical stimulation by unliganded TR, underlining the relative importance of TRE/CRE site in relation to the other TREs in the TRH promoter. [128]
Figure 8. The 5' flanking sequence of the human preproTRH gene between –192 and +58 bp. Four potential thyroid response element (TRE, boxed) and two potential CREB binding elements (CRE, underlined) are shown. One sequence (from –60 to –53 bp) has overlapping TRE/CRE bases (bold). (Modified from Wilber & Xu [128])
![The 5' flanking sequence of the human preproTRH gene between –192 and +58 bp. Four potential thyroid response element (TRE, boxed) and two potential CREB binding elements (CRE, underlined) are shown. One sequence (from –60 to –53 bp) has overlapping TRE/CRE bases (bold). (Modified from Wilber & Xu [128])](./figures4/figure8.gif)
A GRE is also present in TRH gene promoter[113] and glucocorticoid receptor has been identified on TRH neurones of PVN. [137] However, the precise role of corticosteroids in TRH gene expression remains to be elucidated. Both inhibitory and stimulatory effects have been reported. [138, 139] It has been postulated that although the direct effect of glucocorticoids on TRH gene expression is generally stimulatory in vitro, in vivo this activity may be overridden by the complex neuroendocrine reactions following glucocorticoid excess or deficiency. [138] Additional complexity in this field derives from the reports mentioned before of the ACTH releasing inhibitory activity associated to the preproTRH-(178-199) fragment. [119, 120, 140, 141]
Although TRH (either maternal or embryonic) is not required for the normal development of the fetal pituitary thyrotrophs and TRH-deficient mice are not hypothyroid at birth, TRH is required later for the postnatal maintenance of the normal thyrotrophs function. [142]TRH exerts its activity binding to a specific receptor in the plasma membrane of the thyrotroph to induce the release of TSH and to stimulate TSH synthesis. The TRH receptor of several animal species (including humans) has been cloned and has been identified as a G-protein-coupled receptor with seven highly conserved transmembrane domains. [143-145] The presence of inactivating mutations in the 5’-part of TRH receptor gene responsible of congenital central hypothyroidism gas been recently described. [146, 147] TRH-receptor number and mRNA is increased by glucocorticoids and decreased by thyroid hormone as well as by TRH itself. [148, 149] The second messenger for induction of the thyrotroph response to TRH is intracellular Ca2+ ([Ca2+]i). [150-152] TRH was previously believed to act also through stimulation of adenyl cyclase-cAMP pathway, [102] but this result was not confirmed by more recent studies carried out with recombinant TRH-receptor transfected in different cell systems. [153] TRH activates a complex [Ca2+]i response pattern dependent upon both agonist concentration and cell context. The first phase of the TRH response is an acute increase of [Ca2+]i within the thyrotrophs via release from internal stores. This is the consequence of increased inositol triphosphate from hydrolysis of phosphatidyl inositol (PI) in the cell membrane. [150, 154, 155] The hydrolysis of PI is mediated by G protein activation of phospholipase C and also generates diacylglicerol, which in turn activates intracellular protein kinase C (PKC). Stimulation of extracellular calcium influx through verapamil sensitive channels is also observed after TRH stimulation. [152, 155] Both TRH and increased [Ca2+]i stimulate intracellular calcium efflux, which helps in terminating the agonist activity. [155-157] In transfection systems in which the TSH β gene promoter has been linked to a reporter gene, both calcium ionophore ionomycin and phorbol esters (a protein kinase C activator) stimulate TSH gene transcription, confirming the key role of these second messengers in mediating TRH activity. [158] Both increased [Ca2+]i and PKC appear to be independently operative in normal thyrotrophs. [159]
The molecular mechanism(s) underlying the stimulation of TSH β gene expression by TRH have been partially elucidated. In GH3 transfected with hTSH β promoter, two distinct regions of human TSH β gene positively responding to TRH were identified between -130 and +37 bp of the gene. [160-162] (Fig. 4-9) The 3'-region corresponds to eight bp of the first exon; the 5'-region ranged between -128 to -60 bp of the 5'-flanking region. [161, 162] The activity of TRH on TSH β gene promoter is dependent on the presence of other factors such as cAMP response element-binding protein (CREB)-binding protein (CBP). [163, 164] CBP and Pit-1 (see below) also act synergistically with TRH in the stimulation of TSH β gene promoter. [163, 164]
The post-trascriptional effects of TRH on TSH molecule glycosilation and biological activity have been previously discussed.
Thyroid hormone inhibits the effects of TRH on TSH release without interfering with TRH binding to its receptors, but exerting complex negative transcriptional and post-transcriptional activities on TSH synthesis and secretion discussed before.
Thyroid hormone thus regulates TSH secretion by multiple pathways. These are schematically illustrated in Fig. 4-4 and consist of inhibition of the synthesis of TRH receptor in thyrotroph TSH β mRNA as well as of the release of performed, intact TSH. Which specific mechanism is operational in a given physiological situation may depend on the level of circulating thyroid hormones and the duration of the thyroid hormone excess or deficiency. Together, the intricate relationships between thyroid hormone and TSH secretion account for the exquisite sensitivity of measurements of the circulating TSH concentration as a generally reliable indicator of thyroid status.
TRH is rapidly inactivated within the central nervous system by a cell-surface peptidase called TRH-degrading ectoenzyme (TRH-DE) [165]. TRH-DE is very specific, since there is no other ectopeptidase known capable of degrading TRH and TRH is the only known substrate of this unique enzyme[165]. Recently, TRH-DE has been purified to homogeneity and cDNA encoding rat TRH-DE has been cloned. In rodents, pituitary TRH-DE mRNA and enzymatic activity are stringently positively regulated by thyroid hormones, and reduced by estrogens. [165] This suggests that TRH-DE may act as a regulatory element modulating pituitary TSH secretion. The expression of TRH-DE in brain is high and displays a distinct distribution pattern, but it is not influenced by peripheral hormones, supporting the concept that brain TRH-DE may act as a terminator of TRH signals. [165]
A number of other substances, including ubiquitary and pituitary or thyrotroph-specific transcription factors, hormones, neuropeptides and cytokines influence TSH synthesis and secretion (Table 4-3, Fig. 4-9 and 4-10).
Figure 10. Schematic representation of the main factors interacting in the regulation of TSH synthesis and secretion (DA: dopamine; SS: somatostatin; α-AD: α adrenergic pathways). Red arrows: stimulation; blue arrows: inhibition
Table 3. Predominant Effects of Various Agents on TSH Secretion
|
STIMULATORY |
INHIBITORY |
|---|---|
|
Thyrotropin-releasing hormone (TRH) Prostaglandins (?) α-adrenergic agonists (? Via TRH) Opioids (humans) Arginin-vasopressin (AVP) Glucagon-like peptide 1 (GLP-1) Galanin Leptin Glucocorticoids (in vitro) |
Thyroid hormones and analogues Dopamine Gastrin Opioids (rat) Glucocorticoids (in vivo) Serotonin Cholecystokinin (CCK) Gastrin-releasing peptide (GRP) Vasopressin (AVP) Neuropeptide Y (NPY) Interleukin 1β and 6 Tumor necrosis factor α |
Sequence analysis of the hTSH β promoter reveals three areas with high (75-80%) homology to the consensus sequence for the pituitary-specific transcription factor Pit-1. [160-162, 166] These areas are localized between -128 and -58 bp of the 5'-flanking region. Selective mutation analysis revealed that the integrity of these areas was needed for the stimulatory effect of either TRH of forskolin. [167] Expression of an inactive mutant of Pit-1 decreases TRH stimulation of hTSH β [161] and transfection of Pit-1 in cell lines lacking this factor restores cAMP induction of hTSH β gene. [166] Taken together these results strongly support an important role of Pit-1 in the regulation of hTSH β gene expression. Phosphorilation markedly increases the stimulatory activity of Pit-1 in TSH β gene expression, [167] and TRH stimulates transient phosphorilation of Pit-1 in GH3 pituitary cells. [168]
Further support for a role of Pit-1 in the regulation of TSH β gene expression derives from animal models (dwarf mice) and from clinical syndromes of combined pituitary hormone deficiency (CPHD). [147, 169] Snell and Jackson dwarf mice lack of functioning Pit-1 protein due to a point mutation and a gross structural rearrangement in the Pit-1 gene, respectively. [170, 171] Both species show low serum concentration of GH, prolactin and TSH associated to the loss of somato-, lacto- and thyrotropic pituitary cells. Several Pit-1 point mutations and a deletion of the entire coding sequence have been described in patients with CPHD: the effects on TSH secretion differ with the localization of the mutation, but generally result in central hypothyroidism. [76, 169, 172-177]
Although important, the role of Pit-1 for cell-specific expression of TSH β is not as clear as with the GH and PRL genes. [160, 178] Attention has been focused on thyrotropin-specific transcription factors, including Pit-1 splicing variants. Of those, a variant called Pit-1T (containing a 14 aminoacid insertion in the transactivation domain) is found only in thyrotropic cells expressing TSH β and increases TSH β promoter activity when transfected in non-thyrotropic cells expressing wild type Pit-1. [179, 180] These results suggest that the combination of both Pit-1 and Pit-1T may have a synergistic stimulatory effect of TSH β promoter. [181]
As stated before, the transcription factor AP-1 may be involved in modulating the thyroid hormone regulation of TSH-β gene expression. Accordingly, a potential AP-1 binding site is present between -1 to +6 bp of the TSH-β gene, [160] and the integrity of this site is required for maximal stimulation of hTSH-β gene. [79] Haugen et al. [182] described a new 50 kd thyrotroph-specific protein whose binding together with Pit-1 was needed for optimal basal expression of mouse TSH-β gene protein which was subsequently identified as the transcription factor GATA-2. [183] GATA-2 synergistically with Pit-1 stimulates mouse TSH-β promoter activity and is needed for optimal TSH β gene basal activity. Another pituitary-specific protein (P-Lim), which binds and activates common glycoprotein hormone α subunit promoter, also synergizes with Pit-1 in the transcriptional activation of TSH β genes in mice. [184] Recently, a syndrome of CPHD including central hypothyroidism has been described in family members carrying mutations of the pituitary transcription factor prophet of pit-1 (PROP-1). [147, 169, 185, 186] Similarly to Pit-1, this finding suggests an important role of PROP-1 for the cell-specific expression of TSH-β gene.
Increase of intracellular cAMP stimulate expression of both common α and TSH β subunit genes. [162] The action of cAMP is probably exerted not through a direct binding to a CRE sequence, but promoting Pit-1 phosphorilation. [161, 166] The relevance of CBP and its interaction with Pit-1 on stimulation of TSH-β gene has been already mentioned.145.1,145.2
Steroid hormones including corticosteroids, estrogen and testosterone modulate TSH β gene expression. Dexamethasone in pharmacological doses decreases serum TSH concentrations of TSH in normal subjects[187] and in patients[188] or rats[189] with TSH-secreting pituitary adenomas, but does not significantly change TSH subunits mRNA levels. [189] This suggests that glucocorticoids may act on TSH biosynthesis at a translational or post-translational level. Furthermore, as discussed before for TRH gene, several other neuroendocrine mechanisms may participate in vivo in the modulation of TSH synthesis/secretion by glucocorticoids. In keeping with this concept, it has been recently shown in humans that enhanced hypothalamic somatostatinergic and dopaminergic inhibitory activities are involved in the glucocortocoid-dependent blunting of TSH response to TRH. [190]
Estrogens and testosterone have scanty direct effects on TSH synthesis and secretion in humans. Estrogens mildy reduce α and β TSH subunit mRNA in hypothyroid rats, [191] perhaps interacting with the same response elements involved in thyroid hormone regulation. Testosterone has similar effects, at least in part explained by its peripheral conversion to estrogen. [192]
Somatostatin, the major physiological inhibitor of GH secretion, is also an inhibitor of TSH secretion in rats and humans. [193-195] The physiological relevance of this inhibition is suggested by studies carried out with antibodies to somatostatin whose administration in rats increases serum TSH in basal conditions and after TRH or cold-exposure. [121] Indirect evidence for a physiological role of somatostatin in the regulation of TSH secretion has been obtained in humans by the demonstration that stimulation of the endogenous somatostatin tone by oral glucose inhibits TSH response to TRH. [196] The TSH-inhibiting activity of somatostatin is an acute phenomenon, while long-term treatment with somatostatin analogues does not cause hypothyroidism in man, [197, 198] presumably because the effects of the initial decrease in serum thyroid hormone concentration overrides the inhibitory effects of somatostatin. Somatostatin binds to five distinct types of receptors expressed in the anterior pituitary and brain and differing in binding specificities, molecular weight and linkage to adenyl cyclase. [199] Binding of somatostatin to its receptor causes activation of Gi proteins which in turn inhibit adenylate cyclase. Somatostatin also induces cellular hyperpolarization via modulation of voltage-dependent potassium channels: [200] this mechanism is cAMP-independent and leads to a fall of [CA2+]i by reducing extracellular calcium influx. [201]
In animal models, TSH secretion is affected by other hypothalamic hormones: in particular, corticotropin-releasing hormone (CRH) stimulates TSH secretion in chicken[202] through interaction with CRH-receptor-2 [203] and Melanin-concentrating hormone (MCH) suppresses in vivo and in vitro TSH release in rats. [204]
Neurotransmitters are important direct and indirect modulators in TSH synthesis and secretion. A complex network of neurotransmitters neurons terminates on cells bodies of hypophysiotropic neurons and several neurotransmitters (such as dopamine) are directly released into hypophysial portal blood exerting direct effects on anterior pituitary cells. Furthermore, many dopaminergic, serotoninergic, histaminergic, catecolaminergic, opioidergic and GABAergic systems project from other hypothalamic/brain regions to the hypophysiotropic neurons involved in TSH regulation. These projections are important for a normal TSH circadian rhythm, response to stress and cold exposure, while basal TSH secretion is mainly regulated by intrinsic hypothalamic activity. [205] In spite of the difficulty to precisely identify the relative contributions of different neurotransmitter systems in the regulation of TSH secretion, the role of some of them (particularly dopamine and catecholamines) has been rather well defined.
Dopamine, acting via DA2 class of dopamine receptors, inhibits TSH synthesis and release; similarly to somatostatin, this activity is exerted through a decrease in adenylate cyclase. [206-208] Dopamine also inhibits α and TSH-β subunit mRNA and gene transcription in cultured rat anterior pituitary cells. [58] In contrast with its inhibitory activity at the thyrotrophs level, dopamine at the hypothalamic levels stimulates both TRH and somatostatin release, [209, 210] with opposite effect on TSH secretion.
In contrast to dopamine, adrenergic activation exerts a positive regulation on TSH secretion. Central stimulation of α-adrenergic pathways increases TSH release in rat, presumably through stimulation of TRH secretion; furthermore, α1 adrenergic agonists also enhance TSH release from pituitary cells in vitro by mechanisms which are independent of those activated by TRH. [209-212] It is thought that α-adrenergic activity on thyrotrophs is linked to adenylate cyclase activation since agents increasing intracellular cyclic AMP in these cells can increase TSH release. [213, 214]
Opioids inhibit TSH secretion in rats and this action is blocked by the antagonist naloxone, [215] while in humans appear to exert a stimulatory effect, especially on nocturnal TSH surge. [216] Several other neuropeptides may affect TSH secretion in vivo or in vitro. Colecistokinin (CCK), [217] gastrin-releasing peptide (GRP) [218] and neuropeptide Y (NPY) [219] exert inhibitory effects, while arginin-vasopressin (AVP), [220] glucagon-like peptide-1 (GLP-1) [221], galanin[222] and leptin [223, 224] stimulate TSH secretion. Although the precise physiological role of these peptides remains to be clarified, it has been recently suggested that they may be important in connecting the nutrition status and thyroid function, [225] as discussed in more detail later.
Cytokines have recently been demonstrated to have important effects on TRH or TSH release. Both interleukin 1 β (IL-1β) and tumor necrosis factor α (cachectin) inhibit TSH basal release, [226-229] while no inhibition is observed on TSH response to TRH. [230], and this effect is independent from thyroid hormone uptake or receptor occupancy. At the same time, IL-1β stimulates the release of corticotropin-releasing hormone and activates the hypothalamic-pituitary-adrenal axis. [231] Interleukin-1β is produced in rat thyrotrophs, and this production is markedly increased by bacterial lipopolysaccharide[232, 233]. It could thus reduce TSH secretion by either autocrine or paracrine mechanisms. The IL-1β-dependent cytokine interleukin 6 (IL-6) exerts similar inhibitory effects on TSH secretion. Both IL-1β and IL-6 acutely inhibit TSH release from the thyrotrophs, while IL-1β (but not IL-6) also decreases hypothalamic TRH mRNA and gene expression. [234-236] Both IL-1β and IL-6 stimulate 5’-deiodinase activity in cultured pituitary cells, [237] suggesting that increased intrapituitary T4àT3 conversion may be involved in the inhibitory activity on TSH production. IL-6 is produced by the folliculo-stellate cells of the anterior pituitary[238, 239] and, like IL-1β, may regulate TSH release in a paracrine fashion. [231, 234] As discussed later, increased concentration of circulating pro-inflammatory cytokines are involved in the alterations of hypothalamic-pituitary-thyroid axis observed in non-thyroidal illnesses.
Thus an intricate set of relationships within and outside the central nervous system controls the TRH-producing neurones in the medial basal hypothalamus. Alterations in any of these mechanisms can influence TRH and consequently TSH release (Fig. 4-10 and 4-11). The relative importance in human physiology of these neural pathways, which have been directly studied only in animal models, is unknown.
Figure 11. Role fasting, somatostatin (SS) pathways and leptin on TRH and TSH secretion. POMC: pro-opiomelanocortin; α-MSH: α-melanocyte-stimulating hormone; AGRP: Agouti receptor protein; NPY: neuropetide Y; MC4R: melanocortin 4 receptor; PVN: paraventricular nucleus Red arrows: stimulation; blue arrows: inhibition; for further details, see text.
In additional to the classic negative feed-back of thyroid hormone on TSH and TRH secretion detailed in the above paragraph, evidence is accumulating that pituitary TSH is able to inhibit TRH secretion at hypothalamic level (short feedback) and TSH secretion at pituitary level (ultra short feedback). [240] Early observations of inhibition of TSH secretion by injection of pituitary extracts have been recently corroborated by the demonstration of TSH receptor (together with other pituitary hormone receptors) in the hypothalamus[241, 242] and in the folliculo-stellate cells of the adenohypophysis. [243]The precise physiological role of short and ultra-short feedback in controlling TRH/TSH secretion remains to be elucidated. It may be speculated that they concur in the fine tuning of the homeostatic control and in the generation of the pulsatility of TSH secretion. The possibility that thyroid-stimulating autoantibody of Graves’ disease recognize hypothalamic and pituitary TSH receptors has also been suggested to explain suppressed serum TSH levels in some euthyroid Graves’ patients. [240]