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.     

Endotoxin-induced release of interleukin-6 from rat medial basal hypothalami

Interleukin-6 (IL-6) is an inflammatory cytokine that stimulates T-cell activation and B-cell differentiation. We recently reported that picomolar concentrations of IL-6 stimulated PRL, GH, and LH release in vitro. These data suggested that IL-6 may function as a hypothalamic releasing factor for anterior pituitary hormones. Medial basal hypothalami (MBH) were incubated for 60-90 min in Krebs-Ringer bicarbonate buffer supplemented with 0.025% BSA, and the conditioned medium was assayed for IL-6 concentrations by the 7TD1 cell growth factor assay. It was found that MBH released IL-6 in vitro. Although depolarizing concentrations of K+ (56 mM) did not increase IL-6 release, somatostatin release from the MBH was increased significantly. The bacterial endotoxin lipopolysaccharide (LPS; 1-100 ng/ml) induced significant increases in IL-6 release from the MBH. The presence of IL-6 in the hypothalamus suggested a possible role for this cytokine in the regulation of neuropeptide release; however, the release of somatostatin was not affected by 20 ng/ml IL-6. Comparison studies of neural and neuroendocrine tissues revealed that the anterior and posterior pituitaries released larger amounts of bioactive IL-6 than the MBH or parietal cortex during a 4-h incubation; induction of IL-6 release by endotoxin occurred only in the anterior pituitary and hypothalamus. IL-6 mRNA was detectable in the MBH and anterior pituitary tissue after a 4-h incubation; however, no IL-6 mRNA was detectable in freshly isolated tissues. LPS (100 ng/ml) and (Bu)2cAMP (1 mM) increased IL-6 mRNA accumulation in and IL-6 release from the MBH and anterior pituitary. These data suggest that the MBH synthesizes and releases IL-6 via a nonneuronal source in vitro.     

Corticotropin-releasing factor receptor type 2 messenger ribonucleic acid in rat pituitary: localization and regulation by immune challenge,restraint stress,and glucocorticoids

CRF receptor 2 (CRF R2) has been identified in the rat pituitary. However, the cell types that express the receptor remained to be determined. In the present study, we localized CRF R2 mRNA in gonadotropes of the anterior pituitary. Ribonuclease protection assays of anterior pituitary mRNA further showed that the dominant receptor type is CRF R2alpha. We also demonstrated that the expression of CRF R2 in the pituitary is sensitive to alterations to the hypothalamic-pituitary-adrenal axis as CRF R2 mRNA levels in the anterior pituitary of male rats were significantly decreased 6 h after bacterial endotoxin lipopolysaccharide (LPS) injection or restraint stress. Subcutaneous corticosterone injections also resulted in significant suppression of CRF R2 mRNA levels in the pituitary, suggesting that glucocorticoids are involved in modulating CRF R2 mRNA levels in the pituitary under stress. LPS administration still caused a significant suppression of CRF R2 mRNA levels in the anterior pituitary of adrenalectomized rats. This suggests that one or more additional factors is involved in the regulation of CRF R2 expression in the anterior pituitary. Taken together, these data suggest that CRF R2alpha in the anterior pituitary might be involved in the regulation of gonadal functions under stress.     

Bacterial lipopolysaccharide-induced coordinate downregulation of arginine vasopressin receptor V3 and corticotropin-releasing factor receptor 1 messenger ribonucleic acids in the anterior pituitary of endotoxemic steers

AVP and CRF are potent stimulators of pituitary ACTH secretion in cattle. Actions of AVP and CRF at the anterior pituitary are mediated by AVP receptor V3 (V3) and CRF receptor 1 (CRFR1). The primary objective of these studies was to determine the effect of systemic inflammatory stress on V3 and CRFR1 mRNAs in the bovine anterior pituitary. Holstein steers (n=20) were injected with 200 ng/kg bacterial lipopolysaccharide (LPS) and tissues collected 0, 2, 4, 12, and 24 h later. All animals responded to LPS administration with an increase in body temperature, plasma ACTH, and cortisol (p<0.05). Abundance of anterior pituitary V3 mRNA was decreased at 2, 4, and 12 h following LPS administration (p<0.05) and returned to basal by 24 h. A similar temporal regulation of pituitary CRFR1 mRNA (p<0.05), but not pituitary pro-opiomelanocortin (POMC) mRNA, was observed following LPS administration. Similar downregulation of CRFR1 mRNA was not observed in other brain regions following LPS administration (cerebellum, hypothalamus). Our results indicate that V3 and CRFR1 mRNAs are coordinately downregulated in the anterior pituitary during systemic inflammatory stress. Decreased AVP and CRF receptor expression may help regulate the pituitary-adrenal response to stress.     

Simultaneous changes in central and peripheral components of the hypothalamus-pituitary-thyroid axis in lipopolysaccharide-induced acute illness in mice

During illness, major changes in thyroid hormone metabolism and regulation occur; these are collectively known as non-thyroidal illness and are characterized by decreased serum triiodothyronine (T(3)) and thyroxine (T(4)) without an increase in serum TSH. Whether alterations in the central part of the hypothalamus-pituitary-thyroid (HPT) axis precede changes in peripheral thyroid hormone metabolism instead of vice versa, or occur simultaneously, is presently unknown. We therefore studied the time-course of changes in thyroid hormone metabolism in the HPT axis of mice during acute illness induced by bacterial endotoxin (lipopolysaccharide; LPS).LPS rapidly induced interleukin-1beta mRNA expression in the hypothalamus, pituitary, thyroid and liver. This was followed by almost simultaneous changes in the pituitary (decreased expression of thyroid receptor (TR)-beta2, TSHbeta and 5'-deiodinase (D1) mRNAs), the thyroid (decreased TSH receptor mRNA) and the liver (decreased TRbeta1 and D1 mRNA). In the hypothalamus, type 2 deiodinase mRNA expression was strongly increased whereas preproTRH mRNA expression did not change after LPS. Serum T(3) and T(4) fell only after 24 h.Our results suggested almost simultaneous involvement of the whole HPT axis in the downregulation of thyroid hormone metabolism during acute illness.     

The role of central noradrenergic neurons in the control of thyrotropin secretion in the rat.

To investigate the role played by hypothalamic noradrenaline (NE) in the regulation of TRH-TSH release during tonic and cold activated conditions, drugs and surgical procedures able to interfere with central NE tonus were utilized. The time course of the effect of alpha-methyl-para-tyrosine (alpha-MpT) on basal TSH secretion was followed. The tyrosine hydroxylase (TH) inhibitor was unable to modify TSH plasma levels, whereas NE hypothalamic content decreased beginning with the third hour. The acute release of TSH evoked by cold exposure (CE) was prevented by pretreatment with alpha-MpT 1 h before; when alpha-MpT was followed 40 min later by clonidine, a central noradrenergic stimulating agent, TSH response to cold, previously blocked by the TH inhibitor was restored. Intraventricular injection of 10 micrograms of clonidine hydrochloride in unstimulated rats caused a significant rise of basal TSH levels 3, but not 10 min after the administration. Complex deafferentation of the medial basal hypothalamus (MBH), which destroys all the NE fibers afferent to this area, caused no change of thyrotropin secretion in basal conditions. Deafferented animals did not show any acute increase of TSH in response to CE. The results of this study provide evidence that NE may be the catecholamine (CA) mediating the rise in TSH following CE and that the direct stimulation of central NE receptors can evoke a massive TSH release from the anterior pituitary gland also in basal conditions.     

Modulation by oestrogen of thyroid hormone effects on thyrotrophin gene expression

The role of oestrogen in the regulation of TSH gene expression is unclear. We have examined the effect of administration of oestrogen in the rat on serum TSH, pituitary TSH content and pituitary cytoplasmic concentrations of mRNA encoding the TSH beta and alpha subunits, thus deriving measures of hormone release and synthesis. In addition, we have examined the effect of oestrogen on the binding of tri-iodothyronine (T3) to nuclear receptors in the anterior pituitary. Administration of oestrogen did not affect serum concentrations of TSH in euthyroid or untreated hypothyroid rats, but did augment the effects of T3 (1 and 2 micrograms) on serum TSH in hypothyroid animals 6 h after injection of T3. No influence of oestrogen or of thyroid status on pituitary content of TSH was seen. A marked increase in the concentrations of TSH beta and alpha mRNA in pituitary cytoplasm was found in hypothyroidism, compared with those in the euthyroid state. No effect of oestrogen on TSH mRNA was seen in euthyroid animals but concentrations of TSH beta and alpha mRNA were lower in hypothyroid animals than in vehicle-treated controls. A stimulatory influence of T3 on TSH mRNA was seen 6 h after injection of T3; this stimulation was absent in oestrogen-treated rats. No effect of oestrogen on the action of T3 was evident 72 h after beginning treatment with T3. In addition to effects on serum TSH and TSH mRNA, an increase in the number of pituitary nuclear receptors for T3 was seen after oestrogen treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

The endocrine disrupters butyl benzyl phthalate and bisphenol A increase the expression of progesterone receptor messenger ribonucleic acid in the preoptic area of adult ovariectomized rats

Butyl benzyl phthalate (BBP) and bisphenol A (BPA), so-called endocrine disrupters, are known to mimic the action of estrogens: they are thus liable to influence reproductive functions. Since little is known about their action on gene expression in the adult hypothalamus, we examined the effects of these chemicals on the expression of estrogen-regulated mRNAs, i.e., progesterone receptor (PR) mRNA, preproenkephalin (PPE) mRNA and neurotensin (NT) mRNA, in the hypothalamus and pituitary of adult female rats. Two weeks after ovariectomy, rats were subcutaneously injected with 10 mg BBP, 10 mg BPA, or 10 microg 17 beta-estradiol (E(2)) in sesame oil, or with sesame oil alone as a control. Twenty-four hours after the injection, tissues including the preoptic area (POA), mediobasal hypothalamus (MBH) and anterior pituitary were collected. Northern blot revealed that injection of E(2) resulted in expected changes, i.e., significant increases in PR mRNA in the POA, MBH and anterior pituitary, and in PPE mRNA in the MBH. We also found that injection of BPA significantly increased PR mRNA in the POA and anterior pituitary, while injection of BBP increased PR mRNA in the POA and anterior pituitary, although the increase in the anterior pituitary was not significant. No significant effect of E(2), BPA, or BBP on NT mRNA in the POA was detected. The present study demonstrates that the two endocrine disrupters BPA and BBP can increase the expression of PR mRNA in the POA of adult ovariectomized rats.     

Prolactin in cerebrospinal fluid increases the synthesis and release of hypothalamic gamma-aminobutyric acid

The effect of intracerebroventricularly (i.v.t.)-injected rat prolactin (2 micrograms/rat) on the function of tuberoinfundibular gamma-aminobutyric acid (GABA)ergic neurones was assessed in adult male rats by measuring the activity of glutamic acid decarboxylase (GAD) in the mediobasal hypothalamus (MBH) and the concentrations of GABA in hypophysial portal plasma and in the anterior pituitary gland. Fourteen hours after i.v.t. injection of rat prolactin the activity of GAD in the MBH was significantly (P less than 0.05) increased and it remained elevated for at least 16 h after injection. The mean concentrations of GABA in hypophysial portal plasma and in the anterior pituitary were twice those found in vehicle-treated controls 16 h after administration of rat prolactin; no significant effects were observed at earlier time-periods. A significant (P less than 0.01) and long-lasting decrease in endogenous plasma prolactin concentrations was detected 2 h after the i.v.t. injection of rat prolactin and the concentrations remained suppressed for up to 16 h. The present results are consistent with the concept that the activity of tuberoinfundibular GABAergic neurones is regulated, at least in part, by circulating prolactin. The ability of prolactin to accelerate the synthesis and release of GABA in the MBH might constitute a short loop feedback system by which the hormone regulates its own secretion.     

Chronic local inflammation in mice results in decreased TRH and type 3 deiodinase mRNA expression in the hypothalamic paraventricular nucleus independently of diminished food intake

During illness, changes in thyroid hormone metabolism occur, known as nonthyroidal illness and characterised by decreased serum triiodothyronine (T3) and thyroxine (T4) without an increase in TSH. A mouse model of chronic illness is local inflammation, induced by a turpentine injection in each hind limb. Although serum T3 and T4 are markedly decreased in this model, it is unknown whether turpentine administration affects the central part of the hypothalamus-pituitary-thyroid axis (HPT-axis). We therefore studied thyroid hormone metabolism in hypothalamus and pituitary of mice during chronic inflammation induced by turpentine injection. Using pair-fed controls, we could differentiate between the effects of chronic inflammation per se and the effects of restricted food intake as a result of illness. Chronic inflammation increased interleukin (IL)-1beta mRNA expression in the hypothalamus more rapidly than in the pituitary. This hypothalamic cytokine response was associated with a rapid increase in local D2 mRNA expression. By contrast, no changes were present in pituitary D2 expression. TSHbeta mRNA expression was altered compared with controls. Comparing chronic inflamed mice with pair-fed controls, both preproTSH releasing hormone (TRH) and D3 mRNA expression in the paraventricular nucleus were significantly lower 48 h after turpentine administration. The timecourse of TSHbeta mRNA expression was completely different in inflamed mice compared with pair-fed mice. Turpentine administration resulted in significantly decreased TSHbeta mRNA expression only after 24 h while later in time it was lower in pair-fed controls. In conclusion, central thyroid hormone metabolism is altered during chronic inflammation and this cannot solely be attributed to diminished food intake.     

Effect of hypothyroidism and thyroid hormone replacement in vivo on pituitary cytoplasmic concentrations of thyrotropin-beta and alpha-subunit messenger ribonucleic acids

We have studied the influence of hypothyroidism and thyroid hormone replacement in vivo on rat pituitary cytoplasmic concentrations of TSH beta and alpha-subunit mRNA, measured by cytoplasmic dot hybridization, as well as circulating TSH and pituitary TSH content. Cytoplasmic concentrations of GH, PRL, and LH beta-subunit mRNA were measured in parallel. Marked increases in serum TSH and TSH beta and alpha mRNA were found in hypothyroidism; these changes reversed 72 h after beginning T3 replacement. A more marked effect of hypothyroidism on TSH beta mRNA than on alpha mRNA was evident, suggesting differential control of regulation of the TSH genes. In contrast to a rapid fall in serum TSH after T3 administration, an increase in TSH beta and alpha mRNA was evident at 1 and 6 h, followed by a later fall in TSH mRNA to euthyroid values. Pituitary cytoplasmic concentrations of GH and PRL mRNA were reduced in hypothyroidism and increased after administration of T3; LH beta mRNA was unaffected by hypothyroidism or T3 replacement. The differential effects of thyroid status on TSH, GH, PRL, and LH mRNA indicate specificity of regulation of these anterior pituitary hormone genes.     

Studies on the feedback regulation of gonadotropin concentrations in male rats. (II). Partial characterization of 5 alpha-reductase in the medial basal hypothalamus and anterior pituitary gland

To evaluate the testosterone (T) 5 alpha-reductase activity in medial basal hypothalamus (MBH) and anterior pituitary gland of the adult male rat, whole homogenate of MBH or anterior pituitary glands was incubated with 14C-T in the presence of coenzymes under various experimental conditions. Major metabolites were 5 alpha-dihydrotestosterone (DHT), 5 alpha-androstane-3 alpha (beta), 17 beta-diol(3 alpha(beta)-diol) and delta 4 androstenedione. The activity of 5 alpha-reductase was expressed as the sum of the amount of DHT and 3 alpha(beta)-diol formed after incubation. The metabolites converted from T was separated and identified by thin layer chromatography and confirmed by recrystallization. The anterior pituitary gland possessed about four times higher 5 alpha-reductase activity compared with that in MBH. NADPH was essential for 5 alpha-reductase activity. They existed in microsomal fraction. They had same optimum temperature and almost same optimum pH. The Michaelis constant of the 5 alpha-reductase for T in MBH and anterior pituitary gland was 3.1 X 10(-7) M and 5.6 X 10(-7) M, respectively. These results suggest that 5 alpha-reduced metabolites of T, especially DHT have some physiologically significant roles in not only MBH but also anterior pituitary gland. Furthermore, it is noteworthy that 5 alpha-reductase in the anterior pituitary showed higher activity than that in MBH.     

Inhibition of the type 2 iodothyronine deiodinase underlies the elevated plasma TSH associated with amiodarone treatment

The widely prescribed cardiac antiarrhythmic drug amiodarone (AMIO) and its main metabolite, desethylamiodarone (DEA), have multiple side effects on thyroid economy, including an elevation in serum TSH levels. To study the AMIO effect on TSH, mice with targeted disruption of the type 2 deiodinase gene (D2KO) were treated with 80 mg/kg AMIO for 4 wk. Only wild-type (WT) mice controls developed the expected approximate twofold rise in plasma TSH, illustrating a critical role for D2 in this mechanism. A disruption in the D2 pathway caused by AMIO could interfere with the transduction of the T4 signal, generating less T3 and softening the TSH feedback mechanism. When added directly to sonicates of HEK-293 cells transiently expressing D2, both AMIO and DEA behaved as noncompetitive inhibitors of D2 [IC(50) of >100 μm and ～5 μm, respectively]. Accordingly, D2 activity was significantly decreased in the median eminence and anterior pituitary sonicates of AMIO-treated mice. However, the underlying effect on TSH is likely to be at the pituitary gland given that in AMIO-treated mice the paraventricular TRH mRNA levels (which are negatively regulated by D2-generated T3) were decreased. In contrast, AMIO and DEA both exhibited dose-dependent inhibition of D2 activity and elevation of TSH secretion in intact TαT1 cells, a pituitary thyrotroph cell line used to model the TSH feedback mechanism. In conclusion, AMIO and DEA are noncompetitive inhibitors of D2, with DEA being much more potent, and this inhibition at the level of the pituitary gland contributes to the rise in TSH seen in patients taking AMIO.     

Differential effects of leptin and refeeding on the fasting-induced decrease of pituitary type 2 deiodinase and thyroid hormone receptor beta2 mRNA expression in mice

Profound changes in thyroid hormone metabolism occur in the central part of the hypothalamus-pituitary-thyroid (HPT) axis during fasting. Hypothalamic changes are partly reversed by leptin administration, which decreases during fasting. It is unknown to what extent leptin affects the HPT axis at the level of the pituitary. We, therefore, studied fasting-induced alterations in pituitary thyroid hormone metabolism, as well as effects of leptin administration on these changes. Because refeeding rapidly increased serum leptin, the same parameters were studied after fasting followed by refeeding. Fasting for 24 h decreased serum T(3) and T(4) and pituitary TSHbeta, type 2deiodinase (D2), and thyroid hormone receptor beta2 (TRbeta2) mRNA expression. The decrease in D2 and TRbeta2 mRNA expression was prevented when 20 mug leptin was administered twice during fasting. By contrast, the decrease in TSHbeta mRNA expression was unaffected. A single dose of leptin given after 24 h fasting did not affect decreased TSHbeta, D2, and TRbeta2 mRNA expression, while 4 h refeeding resulted in pituitary D2 and TRbeta2 mRNA expression as observed in control mice. Serum leptin, T(3), and T(4) after refeeding were similar compared with leptin administration. We conclude that fasting decreases pituitary TSHbeta, D2, and TRbeta2 mRNA expression, which (with the exception of TSHbeta) can be prevented by leptin administration during fasting. Following 24 h fasting, 4 h refeeding completely restores pituitary D2 and TRbeta2 mRNA expression, while a single leptin dose is ineffective. This indicates that other postingestion signals may be necessary to modulate rapidly the fasting-induced decrease in pituitary D2 and TRbeta2 mRNA expression.     

Vasoactive intestinal peptide (VIP) mediates the effect of estrogens on the dopaminergic tone in the hypothalamic-pituitary axis of ovariectomized (OVX) rats

The role of vasoactive intestinal peptide (VIP) in the regulation of dopamine (DA) concentration in mediobasal hypothalamus (MBH), posterior and anterior pituitary of ovariectomized (OVX) estrogenized rats was studied using passive immunization against VIP with a specific antiserum (a-VIP). Chronic estradiol administration decreased DA concentration in MBH, and in posterior and anterior pituitary, compared to OVX control rats. DA tissue concentration increased following a-VIP administration to control and estrogenized OVX rats. In vitro study of VIP and a-VIP on DA release from MBH in chronically estrogenized OVX rats showed that estrogens decreased DA evoked-release from MBH; a-VIP increased DA evoked-release from MBH of control OVX and estrogenized rats. VIP decreased DA evoked-release from MBH of OVX rats, but had no effect on estrogenized rats. VIP decreased DA tissue concentration in MBH of OVX control but not of estrogenized rats. It is suggested that VIP decreases DA synthesis and release from hypothalamic neurons in female rats, and that VIP partially mediates the inhibitory effect of long-term estrogen administration on DA release from MBH.     

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.