Kristen R. Vella

NPY and MC4R Signaling Regulate Thyroid Hormone Levels during Fasting through Both Central and Peripheral Pathways

Fasting-induced suppression of the hypothalamic-pituitary-thyroid (HPT) axis is an adaptive response to decrease energy expenditure during food deprivation. Previous studies demonstrate that leptin communicates nutritional status to the HPT axis through thyrotropin-releasing hormone (TRH) in the paraventricular nucleus (PVN) of the hypothalamus. Leptin targets TRH neurons either directly or indirectly via the arcuate nucleus through pro-opiomelanocortin (POMC) and agouti-related peptide/neuropeptide Y (AgRP/NPY) neurons. To evaluate the role of these pathways in vivo, we developed double knockout mice that lack both the melanocortin 4 receptor (MC4R) and NPY. We show that NPY is required for fasting-induced suppression of Trh expression in the PVN. However, both MC4R and NPY are required for activation of hepatic pathways that metabolize T(4) during the fasting response. Thus, these signaling pathways play a key role in the communication of fasting signals to reduce thyroid hormone levels both centrally and through a peripheral hepatic circuit.

Regulation of thyrotropin-releasing hormone-expressing neurons in paraventricular nucleus of the hypothalamus by signals of adiposity

Fasting-induced suppression of thyroid hormone levels is an adaptive response to reduce energy expenditure in both humans and mice. This suppression is mediated by the hypothalamic-pituitary-thyroid axis through a reduction in TRH levels expressed in neurons of the paraventricular nucleus of the hypothalamus (PVN). TRH gene expression is positively regulated by leptin. Whereas decreased leptin levels during fasting lead to a reduction in TRH gene expression, the mechanisms underlying this process are still unclear. Indeed, evidence exists that TRH neurons in the PVN are targeted by leptin indirectly via the arcuate nucleus, whereas correlative evidence for a direct action exists as well. Here we provide both in vivo and in vitro evidence that the activity of hypothalamic-pituitary-thyroid axis is regulated by both direct and indirect leptin regulation. We show that both leptin and α-MSH induce significant neuronal activity mediated through a postsynaptic mechanism in TRH-expressing neurons of PVN. Furthermore, we provide in vivo evidence indicating the contribution of each pathway in maintaining serum levels of thyroid hormone.

The Thyrotropin-Releasing Hormone Gene Is Regulated by Thyroid Hormone at the Level of Transcription in Vivo

The expression of the TRH gene in the paraventricular nucleus (PVH) of the hypothalamus is required for the normal production of thyroid hormone (TH) in rodents and humans. In addition, the regulation of TRH mRNA expression by TH, specifically in the PVH, ensures tight control of the set point of the hypothalamic-pituitary-thyroid axis. Although many studies have assumed that the regulation of TRH expression by TH is at the level of transcription, there is little data available to demonstrate this. We used two in vivo model systems to show this. In the first model system, we developed an in situ hybridization (ISH) assay directed against TRH heteronuclear RNA to measure TRH transcription directly in vivo. We show that in the euthyroid state, TRH transcription is present both in the PVH and anterior/lateral hypothalamus. In the hypothyroid state, transcription is activated in the PVH only and can be shut off within 5 h by TH. In the second model system, we employed transgenic mice that express the Cre recombinase under the control of the genomic region containing the TRH gene. Remarkably, TH regulates Cre expression in these mice in the PVH only. Taken together, these data affirm that TH regulates TRH at the level of transcription in the PVH only and that genomic elements surrounding the TRH gene mediate its regulation by T(3). Thus, it should be possible to identify the elements within the TRH locus that mediate its regulation by T(3) using in vivo approaches.

Thyroid hormone signaling in vivo requires a balance between coactivators and corepressors.

ABSTRACT Resistance to thyroid hormone (RTH), a human syndrome, is characterized by high thyroid hormone (TH) and thyroid-stimulating hormone (TSH) levels. Mice with mutations in the thyroid hormone receptor beta (TRβ) gene that cannot bind steroid receptor coactivator 1 (SRC-1) and Src-1−/− mice both have phenotypes similar to that of RTH. Conversely, mice expressing a mutant nuclear corepressor 1 (Ncor1) allele that cannot interact with TRβ, termed NCoRΔID, have low TH levels and normal TSH. We hypothesized that Src-1−/− mice have RTH due to unopposed corepressor action. To test this, we crossed NCoRΔID and Src-1−/− mice to create mice deficient for coregulator action in all cell types. Remarkably, NCoRΔID/ΔID Src-1−/− mice have normal TH and TSH levels and are triiodothryonine (T3) sensitive at the level of the pituitary. Although absence of SRC-1 prevented T3 activation of key hepatic gene targets, NCoRΔID/ΔID Src-1−/− mice reacquired hepatic T3 sensitivity. Using in vivo chromatin immunoprecipitation assays (ChIP) for the related coactivator SRC-2, we found enhanced SRC-2 recruitment to TR-binding regions of genes in NCoRΔID/ΔID Src-1−/− mice, suggesting that SRC-2 is responsible for T3 sensitivity in the absence of NCoR1 and SRC-1. Thus, T3 targets require a critical balance between NCoR1 and SRC-1. Furthermore, replacement of NCoR1 with NCoRΔID corrects RTH in Src-1−/− mice through increased SRC-2 recruitment to T3 target genes.

The selective loss of the type 2 iodothyronine deiodinase in mouse thyrotrophs increases basal TSH but blunts the thyrotropin response to hypothyroidism

The type 2 iodothyronine deiodinase (D2) is essential for feedback regulation of TSH by T4. We genetically inactivated in vivo D2 in thyrotrophs using a mouse model of Cga-driven cre recombinase. Pituitary D2 activity was reduced 90% in the Cga-cre D2 knockout (KO) mice compared with control Dio2(fl/fl) mice. There was no growth or reproductive phenotype. Basal TSH levels were increased 1.5- to 1.8-fold, but serum T4 and T3 were not different from the controls in adult mice. In hypothyroid adult mice, suppression of TSH by T4, but not T3, was impaired. Despite mild basal TSH elevation, the TSH increase in response to hypothyroidism was 4-fold reduced in the Cga-cre D2KO compared with control mice despite an identical level of pituitary TSH α- and β-subunit mRNAs. In neonatal Cga-cre D2KO mice, TSH was also 2-fold higher than in the controls, but serum T4 was elevated. Despite a constant TSH, serum T4 increased 2-3-fold between postnatal day (P) 5 and P15 in both genotypes. The pituitary, but not cerebrocortical, D2 activity was markedly elevated in P5 mice decreasing towards adult levels by P17. In conclusion, a congenital severe reduction of thyrotroph D2 causes a major impairment of the TSH response to hypothyroidism. This would be deleterious to the compensatory adaptation of the thyroid gland to iodine deficiency.

The Ups and Downs of Thyrotropin-Releasing Hormone

The synthesis and secretion of thyrotropin-releasing hormone (TRH) is absolutely required for intact thyroid function in humans and rodents. Furthermore, feedback regulation of TRH production by thyroid hormone allows for tight maintenance of circulating thyroid hormone levels and thus establishes the hypothalamic-pituitary-thyroid (H-P-T) axis. Although the TRH gene is expressed in many regions of the brain, its regulation by thyroid hormone is restricted to neurons in the paraventricular nucleus of the hypothalamus (PVH) (1). This discrete set of neurons in the PVH, termed hypophysiotropic, project to the median eminence where mature TRH peptide is released into the portal capillary system bound for TRH-receptors present in the anterior pituitary (Fig. 1​1).). Whereas it is clear that thyroid hormone regulates TRH production in the PVH both at the level of gene expression and posttranslational processing, in this issue of Endocrinology, Sanchez et al. (2) provide evidence that thyroid hormone also controls TRH peptide degradation by regulating the enzyme pyroglutamyl peptidase II (PPII). This enzyme is expressed in tanycytes; glial cells that line the third ventricle in the hypothalamus whose cytoplasmic processes extend into the median eminence. Thus, PPII appears to regulate TRH peptide bioavailability upstream of the pituitary.

Age-Related Hearing Loss and Degeneration of Cochlear Hair Cells in Mice Lacking Thyroid Hormone Receptor β1.

A key function of the thyroid hormone receptor β (Thrb) gene is in the development of auditory function. However, the roles of the 2 receptor isoforms, TRβ1 and TRβ2, expressed by the Thrb gene are unclear, and it is unknown whether these isoforms promote the maintenance as well as development of hearing. We investigated the function of TRβ1 in mice with a Thrb(b1) reporter allele that expresses β-galactosidase instead of TRβ1. In the immature cochlea, β-galactosidase was detected in the greater epithelial ridge, sensory hair cells, spiral ligament, and spiral ganglion and in adulthood, at low levels in the hair cells, support cells and root cells of the outer sulcus. Although deletion of all TRβ isoforms causes severe, early-onset deafness, deletion of TRβ1 or TRβ2 individually caused no obvious hearing loss in juvenile mice. However, over subsequent months, TRβ1 deficiency resulted in progressive loss of hearing and loss of hair cells. TRβ1-deficient mice had minimal changes in serum thyroid hormone and thyrotropin levels, indicating that hormonal imbalances were unlikely to cause hearing loss. The results suggest mutually shared roles for TRβ1 and TRβ2 in cochlear development and an unexpected requirement for TRβ1 in the maintenance of hearing in adulthood.

PITX2 AND PITX1 Regulate Thyrotroph Function and Response to Hypothyroidism

Pitx2 is a homeodomain transcription factor required in a dose-dependent manner for the development of multiple organs. Pitx2-null homozygotes (Pitx2(-/-)) have severe pituitary hypoplasia, whereas mice with reduced-function alleles (Pitx2(neo/neo)) exhibit modest hypoplasia and reduction in the developing gonadotroph and Pou1f1 lineages. PITX2 is expressed broadly in Rathkes pouch and the fetal pituitary gland. It predominates in adult thyrotrophs and gonadotrophs, although it is not necessary for gonadotroph function. To test the role of PITX2 in thyrotroph function, we developed thyrotroph-specific cre transgenic mice, Tg(Tshb-cre) with a recombineered Tshb bacterial artificial chromosome that ablates floxed genes in differentiated pituitary thyrotrophs. We used the best Tg(Tshb-Cre) strain to generate thyrotroph-specific Pitx2-deficient offspring, Pitx2(flox/-;)Tg(Tshb-cre). Double immunohistochemistry confirmed Pitx2 deletion. Pitx2(flox/-);Tg(Tshb-cre) mice have a modest weight decrease. The thyroid glands are smaller, although circulating T(4) and TSH levels are in the normal range. The pituitary levels of Pitx1 transcripts are significantly increased, suggesting a compensatory mechanism. Hypothyroidism induced by low-iodine diet and oral propylthiouracil revealed a blunted TSH response in Pitx2(flox/-);Tg(Tshb-cre) mice. Pitx1 transcripts increased significantly in control mice with induced hypothyroidism, but they remained unchanged in Pitx2(flox/-);Tg(Tshb-cre) mice, possibly because Pitx1 levels were already maximally elevated in untreated mutants. These results suggest that PITX2 and PITX1 have overlapping roles in thyrotroph function and response to hypothyroidism. The novel cre transgene that we report will be useful for studying the function of other genes in thyrotrophs.

Family members CREB and CREM control thyrotropin-releasing hormone (TRH) expression in the hypothalamus

Thyrotropin-releasing hormone (TRH) in the paraventricular nucleus (PVN) of the hypothalamus is regulated by thyroid hormone (TH). cAMP response element binding protein (CREB) has also been postulated to regulate TRH expression but its interaction with TH signaling in vivo is not known. To evaluate the role of CREB in TRH regulation in vivo, we deleted CREB from PVN neurons to generate the CREB1(ΔSIM1) mouse. As previously shown, loss of CREB was compensated for by an up-regulation of CREM in euthyroid CREB1(ΔSIM1) mice but TSH, T₄ and T₃ levels were normal, even though TRH mRNA levels were elevated. Interestingly, TRH mRNA expression was also increased in the PVN of CREB1(ΔSIM1) mice in the hypothyroid state but became normal when made hyperthyroid. Importantly, CREM levels were similar in CREB1(ΔSIM1) mice regardless of thyroid status, demonstrating that the regulation of TRH by T₃ in vivo likely occurs independently of the CREB/CREM family.

Mice With Hepatocyte-Specific Deficiency of Type 3 Deiodinase Have Intact Liver Regeneration and Accelerated Recovery From Nonthyroidal Illness After Toxin-Induced Hepatonecrosis

Type 3 deiodinase (D3), the physiologic inactivator of thyroid hormones, is induced during tissue injury and regeneration. This has led to the hypotheses that D3 impacts injury tolerance by reducing local T3 signaling and contributes to the fall in serum triiodothyronine (T3) observed in up to 75% of sick patients (termed the low T3 syndrome). Here we show that a novel mutant mouse with hepatocyte-specific D3 deficiency has normal local responses to toxin-induced hepatonecrosis, including normal degrees of tissue necrosis and intact regeneration, but accelerated systemic recovery from illness-induced hypothyroxinemia and hypotriiodothyroninemia, demonstrating that peripheral D3 expression is a key modulator of the low T3 syndrome.