The Musashi-1-Type 2 Deiodinase Pathway Regulates Astrocyte Proliferation.

Thyroid hormone (TH) is a critical regulator of cellular function and cell fate. The circulating TH level is relatively stable while tissue TH action fluctuates according to cell-type specific mechanisms. Here we focused on identifying mechanisms that regulate TH action through the type 2 deiodinase (D2) in glial cells. Dio2 mRNA has an unusually long 3' untranslated region (3'UTR) where we identified multiple putative MSI1 binding sites for Musashi-1 (MSI1), a highly conserved RNA-binding cell cycle regulator. Binding to these sites was confirmed through electrophoretic mobility shift assay. In H4 glioma cells, shRNA-mediated MSI1 knockdown increased endogenous D2 activity, whereas MSI1 overexpression in HEK293T cells decreased D2 expression. This latter effect could be prevented by the deletion of a 3.6 kb region of the 3'UTR of Dio2 mRNA containing MSI1 binding sites. MSI1-immunoreactivity was observed in two mouse Dio2-expressing cell types, i.e. cortical astrocytes and hypothalamic tanycytes, establishing the anatomical basis for a potential in vivo interaction of Dio2 mRNA and MSl1. Indeed, increased D2 expression was observed in the cortex of mice lacking MSI1 protein. Furthermore, MSI1 knockdown-induced D2 expression slowed down cell proliferation by 56% in primary cultures of mouse cortical astrocytes, establishing the functionality of the MSI1-D2-T3 pathway. In summary, Dio2 mRNA is a target of MSI1 and the MSI1-D2-T3 pathway is a novel regulatory mechanism of astrocyte proliferation with the potential to regulate the pathogenesis of human glioblastoma.

Unique, Specific CART Receptor-Independent Regulatory Mechanism of CART(55-102) Peptide in Spinal Nociceptive Transmission and Its Relation to Dipeptidyl-Peptidase 4 (DDP4).

Cocaine- and amphetamine-regulated transcript (CART) peptides are involved in several physiological and pathological processes, but their mechanism of action is unrevealed due to the lack of identified receptor(s). We provided evidence for the antihyperalgesic effect of CART(55-102) by inhibiting dipeptidyl-peptidase 4 (DPP4) in astrocytes and consequently reducing neuroinflammation in the rat spinal dorsal horn in a carrageenan-evoked inflammation model. Both naturally occurring CART(55-102) and CART(62-102) peptides are present in the spinal cord. CART(55-102) is not involved in acute nociception but regulates spinal pain transmission during peripheral inflammation. While the full-length peptide with a globular motif contributes to hyperalgesia, its N-terminal inhibits this process. Although the anti-hyperalgesic effects of CART(55-102), CART(55-76), and CART(62-76) are blocked by opioid receptor antagonists in our inflammatory models, but not in neuropathic Seltzer model, none of them bind to any opioid or G-protein coupled receptors. DPP4 interacts with Toll-like receptor 4 (TLR4) signalling in spinal astrocytes and enhances the TLR4-induced expression of interleukin-6 and tumour necrosis factor alpha contributing to inflammatory pain. Depending on the state of inflammation, CART(55-102) is processed in the spinal cord, resulting in the generation of biologically active isoleucine-proline-isoleucine (IPI) tripeptide, which inhibits DPP4, leading to significantly decreased glia-derived cytokine production and hyperalgesia.

Tetrabromobisphenol A and Diclazuril Evoke Tissue-Specific Changes of Thyroid Hormone Signaling in Male Thyroid Hormone Action Indicator Mice.

Thyroid hormone (TH) signaling is a prerequisite of normal tissue function. Environmental pollutants with the potential to disrupt endocrine functions represent an emerging threat to human health and agricultural production. We used our Thyroid Hormone Action Indicator (THAI) mouse model to study the effects of tetrabromobisphenol A (TBBPA; 150 mg/bwkg/day orally for 6 days) and diclazuril (10.0 mg/bwkg/day orally for 5 days), a known and a potential hormone disruptor, respectively, on local TH economy. Tissue-specific changes of TH action were assessed in 90-day-old THAI mice by measuring the expression of a TH-responsive luciferase reporter in tissue samples and by in vivo imaging (14-day-long treatment accompanied with imaging on day 7, 14 and 21 from the first day of treatment) in live THAI mice. This was followed by promoter assays to elucidate the mechanism of the observed effects. TBBPA and diclazuril impacted TH action differently and tissue-specifically. TBBPA disrupted TH signaling in the bone and small intestine and impaired the global TH economy by decreasing the circulating free T4 levels. In the promoter assays, TBBPA showed a direct stimulatory effect on the hdio3 promoter, indicating a potential mechanism for silencing TH action. In contrast, diclazuril acted as a stimulator of TH action in the liver, skeletal muscle and brown adipose tissue without affecting the Hypothalamo-Pituitary-Thyroid axis. Our data demonstrate distinct and tissue-specific effects of TBBPA and diclazuril on local TH action and prove that the THAI mouse is a novel mammalian model to identify TH disruptors and their tissue-specific effects.

Hepatic Inactivation of the Type 2 Deiodinase Confers Resistance to Alcoholic Liver Steatosis.

BACKGROUND: A mouse with hepatocyte-specific deiodinase type II inactivation (Alb-D2KO) is resistant to diet-induced obesity, hepatic steatosis, and hypertriglyceridemia due to perinatal epigenetic modifications in the liver. This phenotype is linked to low levels of Zfp125, a hepatic transcriptional repressor that promotes liver steatosis by inhibiting genes involved in packaging and secretion of very-low-density lipoprotein. METHODS: Here, we used chronic and binge ethanol (EtOH) in mice to cause liver steatosis. RESULTS: The EtOH treatment causes a 2.3-fold increase in hepatic triglyceride content; Zfp125 levels were approximately 50% higher in these animals. In contrast, Alb-D2KO mice did not develop EtOH-induced liver steatosis. They also failed to elevate Zfp125 to the same levels, despite being on the EtOH-containing diet for the same period of time. Their phenotype was associated with 1.3- to 2.9-fold up-regulation of hepatic genes involved in lipid transport and export that are normally repressed by Zfp125, that is, Mttp, Abca1, Ldlr, Apoc1, Apoc3, Apoe, Apoh, and Azgp1. Furthermore, genes involved in the EtOH metabolic pathway, that is, Aldh2 and Acss2, were also 1.6- to 3.1-fold up-regulated in Alb-D2KO EtOH mice compared with control animals kept on EtOH. CONCLUSIONS: EtOH consumption elevates expression of Zfp125. Alb-D2KO animals, which have lower levels of Zfp125, are much less susceptible to EtOH-induced liver steatosis.

Perinatal deiodinase 2 expression in hepatocytes defines epigenetic susceptibility to liver steatosis and obesity.

Thyroid hormone binds to nuclear receptors and regulates gene transcription. Here we report that in mice, at around the first day of life, there is a transient surge in hepatocyte type 2 deiodinase (D2) that activates the prohormone thyroxine to the active hormone triiodothyronine, modifying the expression of ∼165 genes involved in broad aspects of hepatocyte function, including lipid metabolism. Hepatocyte-specific D2 inactivation (ALB-D2KO) is followed by a delay in neonatal expression of key lipid-related genes and a persistent reduction in peroxisome proliferator-activated receptor-γ expression. Notably, the absence of a neonatal D2 peak significantly modifies the baseline and long-term hepatic transcriptional response to a high-fat diet (HFD). Overall, changes in the expression of approximately 400 genes represent the HFD response in control animals toward the synthesis of fatty acids and triglycerides, whereas in ALB-D2KO animals, the response is limited to a very different set of only approximately 200 genes associated with reverse cholesterol transport and lipase activity. A whole genome methylation profile coupled to multiple analytical platforms indicate that 10-20% of these differences can be related to the presence of differentially methylated local regions mapped to sites of active/suppressed chromatin, thus qualifying as epigenetic modifications occurring as a result of neonatal D2 inactivation. The resulting phenotype of the adult ALB-D2KO mouse is dramatic, with greatly reduced susceptibility to diet-induced steatosis, hypertriglyceridemia, and obesity.

Thyroid hormone activation by type 2 deiodinase mediates exercise-induced peroxisome proliferator-activated receptor-γ coactivator-1α expression in skeletal muscle.

KEY POINTS: In skeletal muscle, physical exercise and thyroid hormone mediate the peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1a) expression that is crucial to skeletal muscle mitochondrial function. The expression of type 2 deiodinase (D2), which activates thyroid hormone in skeletal muscle is upregulated by acute treadmill exercise through a ß-adrenergic receptor-dependent mechanism. Pharmacological block of D2 or disruption of the Dio2 gene in skeletal muscle fibres impaired acute exercise-induced PGC-1a expression. Dio2 disruption also impaired muscle PGC-1a expression and mitochondrial citrate synthase activity in chronically exercised mice. ABSTRACT: Thyroid hormone promotes expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1a), which mediates mitochondrial biogenesis and oxidative capacity in skeletal muscle (SKM). Skeletal myocytes express the type 2 deiodinase (D2), which generates 3,5,3'-triiodothyronine (T3 ), the active thyroid hormone. To test whether D2-generated T3 plays a role in exercise-induced PGC-1a expression, male rats and mice with SKM-specific Dio2 inactivation (SKM-D2KO or MYF5-D2KO) were studied. An acute treadmill exercise session (20 min at 70-75% of maximal aerobic capacity) increased D2 expression/activity (1.5- to 2.7-fold) as well as PGC-1a mRNA levels (1.5- to 5-fold) in rat soleus muscle and white gastrocnemius muscle and in mouse soleus muscle, which was prevented by pretreatment with 1 mg (100 g body weight)(-1) propranolol or 6 mg (100 g body weight)(-1) iopanoic acid (5.9- vs. 2.8-fold; P < 0.05), which blocks D2 activity . In the SKM-D2KO mice, acute treadmill exercise failed to induce PGC-1a fully in soleus muscle (1.9- vs. 2.8-fold; P < 0.05), and in primary SKM-D2KO myocytes there was only a limited PGC-1a response to 1 µm forskolin (2.2- vs. 1.3-fold; P < 0.05). Chronic exercise training (6 weeks) increased soleus muscle PGC-1a mRNA levels (∼25%) and the mitochondrial enzyme citrate synthase (∼20%). In contrast, PGC-1a expression did not change and citrate synthase decreased by ∼30% in SKM-D2KO mice. The soleus muscle PGC-1a response to chronic exercise was also blunted in MYF5-D2KO mice. In conclusion, acute treadmill exercise increases SKM D2 expression through a ß-adrenergic receptor-dependent mechanism. The accelerated conversion of T4 to T3 within myocytes mediates part of the PGC-1a induction by treadmill exercise and its downstream effects on mitochondrial function.

GLP-1 Receptor Signaling Has Different Effects on the Perikarya and Axons of the Hypophysiotropic Thyrotropin-Releasing Hormone Synthesizing Neurons in Male Mice.

Background: Glucagon-like peptide 1 (GLP-1) is involved in the regulation of energy and glucose homeostasis. As GLP-1 has similar effects on the energy homeostasis as the hypophysiotropic thyrotropin-releasing hormone (TRH) neurons that regulate the hypothalamic-pituitary-thyroid (HPT) axis, we raised the possibility that the TRH neurons are involved in the mediation of the effects of GLP-1. Therefore, the relationship and interaction of the GLP-1 system and the TRH neurons of the hypothalamic paraventricular nucleus (PVN) were studied. Methods: To examine the anatomical and functional relationship of TRH neurons and the GLP-1 system in the PVN, immunocytochemistry, in situ hybridization, in vitro patch-clamp electrophysiology, metabolic phenotyping, and explant experiments were performed. Results: Our data demonstrate that the TRH neurons of the PVN are innervated by GLP-1 producing neurons and express the GLP-1 receptor (GLP-1R). However, not only do the GLP-1-innervated TRH neurons express GLP-1R but the receptor is also present in the axons of the hypophysiotropic TRH neurons in the blood-brain barrier free median eminence (ME) suggesting that peripherally derived GLP-1 may also influence the TRH neurons. In vitro, GLP-1 increased the firing rate of TRH neurons and depolarized them. In addition, GLP-1 directly stimulated the GABAergic input of a population of TRH neurons. Furthermore, GLP-1 inhibited the release of TRH from the hypophysiotropic axons in the ME. In vivo, peripheral GLP-1R agonist administration markedly inhibited the food intake and the energy expenditure, but had no effect on the TRH expression in the PVN and resulted in lower circulating free T4 levels. Conclusions: Our results indicate that GLP-1R activation has a direct stimulatory effect on TRH neurons in the PVN, but the activation of GLP-1R may also inhibit TRH neurons by facilitating their inhibitory inputs or by inhibiting the axon terminals of these cells in the ME. The innervation of TRH neurons by GLP-1 neurons suggests that TRH neurons might be influenced by both circulating GLP-1 and by GLP-1 neurons of the nucleus tractus solitarii. The lack of GLP-1R agonist-induced regulation of TRH neurons in vivo suggests that the HPT axis does not mediate the GLP-1R agonist-induced weight loss.

The Perioperative Period of Heart Transplantation Is Affected by Thyroid Hormone Status.

Background: Orthotopic heart transplantation (HTx) is a long-term surgical therapeutic approach for patients with end-stage heart failure. The objective of the present study was to uncover associations between altered thyroid hormone (TH) status and adverse outcomes after HTx. Methods: In this prospective, single-center cohort study, 283 patients underwent HTx between 2013 and 2020 at the Heart and Vascular Center of Semmelweis University in Hungary. We measured serum free triiodothyronine (fT3), free thyroxine (fT4), and thyrotropin (TSH) pre- and postoperatively. TaqMan qPCR was used to measure type 2 deiodinase and type 3 deiodinase mRNA (Dio2 and Dio3, respectively) levels from the diseased heart bioptates. To assess the local TH action of the heart, mRNA levels of Hcn2 and Myh7 were measured in a subgroup of patients receiving extracorporeal membrane oxygenation (ECMO) postoperatively. Groups were compared using nonparametric tests. Cox regression analysis and logistic regression test were used to investigate the outcomes. The connection between serum TH parameters and cardiac gene expressions was assessed using linear regression. Results: Serum TSH (p = 0.009), fT3 (p < 0.001), and fT4 (p < 0.001) levels were lower after HTx than preoperatively. Levothyroxine (LT4) administered to donors was associated with better survival after 30 days (p = 0.049). LT4 replacement given to recipients after HTx was associated with better survival after 30 days (p = 0.018), 1 year (p = 0.002), and 2 years (p = 0.001). Dio3 mRNA level was significantly increased in patients who were treated with ECMO (p = 0.026), left ventricular assist device (LVAD) (p = 0.008), and biventricular assist device (BiVAD) (p = 0.013) preoperatively, and ECMO (p = 0.042) postoperatively, compared with those who did not require any type of mechanical circulatory support (MCS). We found no significant difference in the expression of the Hcn2 and Myh7 marker genes between patients on postoperative ECMO and those without MCS, and neither did they correlate with serum hormone levels (p = 0.519 and p = 0.056, respectively). Conclusions: We conclude that TH status plays an important role in HTx patients, and monitoring of TH status in the perioperative period may contribute to improved treatment outcomes. Our findings require independent confirmation in a randomized controlled clinical trial.

Topography of the GLP-1/GLP-1 receptor system in the spinal cord of male mice.

Glucagon-like peptide-1 receptor (GLP-1R) agonists are now commonly used to treat type 2 diabetes and obesity. GLP-1R signaling in the spinal cord has been suggested to account for the mild tachycardia caused by GLP-1R agonists, and may also be involved in the therapeutic effects of these drugs. However, the neuroanatomy of the GLP-1/GLP-1R system in the spinal cord is still poorly understood. Here we applied in situ hybridization and immunohistochemistry to characterize this system, and its relation to cholinergic neurons. GLP-1R transcript and protein were expressed in neuronal cell bodies across the gray matter, in matching distribution patterns. GLP-1R-immunolabeling was also robust in dendrites and axons, especially in laminae II-III in the dorsal horn. Cerebrospinal fluid-contacting neurons expressed GLP-1R protein at exceedingly high levels. Only small subpopulations of cholinergic neurons expressed GLP-1R, including a subset of sympathetic preganglionic neurons at the rostral tip of the intermediolateral nucleus. GLP-1 axons innervated all regions where GLP-1R neurons were distributed, except laminae II-III. Scattered preproglucagon (Gcg) mRNA-expressing neurons were identified in the cervical and lumbar enlargements. The results will facilitate further studies on how GLP-1 regulates the sympathetic system and other autonomic and somatic functions via the spinal cord.

Neuronal hypoxia induces Hsp40-mediated nuclear import of type 3 deiodinase as an adaptive mechanism to reduce cellular metabolism.

In neurons, the type 3 deiodinase (D3) inactivates thyroid hormone and reduces oxygen consumption, thus creating a state of cell-specific hypothyroidism. Here we show that hypoxia leads to nuclear import of D3 in neurons, without which thyroid hormone signaling and metabolism cannot be reduced. After unilateral hypoxia in the rat brain, D3 protein level is increased predominantly in the nucleus of the neurons in the pyramidal and granular ipsilateral layers, as well as in the hilus of the dentate gyrus of the hippocampal formation. In hippocampal neurons in culture as well as in a human neuroblastoma cell line (SK-N-AS), a 24 h hypoxia period redirects active D3 from the endoplasmic reticulum to the nucleus via the cochaperone Hsp40 pathway. Preventing nuclear D3 import by Hsp40 knockdown resulted an almost doubling in the thyroid hormone-dependent glycolytic rate and quadrupling the transcription of thyroid hormone target gene ENPP2. In contrast, Hsp40 overexpression increased nuclear import of D3 and minimized thyroid hormone effects in cell metabolism. In conclusion, ischemia/hypoxia induces an Hsp40-mediated translocation of D3 to the nucleus, facilitating thyroid hormone inactivation proximal to the thyroid hormone receptors. This adaptation decreases thyroid hormone signaling and may function to reduce ischemia-induced hypoxic brain damage.

In vivo Characterization of Endocrine Disrupting Chemical Effects via Thyroid Hormone Action Indicator Mouse.

Thyroid hormones (TH) play a critical role in cell metabolism and tissue function. TH economy is susceptible to endocrine disrupting chemicals (EDCs) that can disturb hormone production or action. Many environmental pollutants are EDCs, representing an emerging threat to both human health and agricultural production. This has led to an increased demand for proper test systems to examine the effects of potential EDCs. However, current methodologies face challenges. Most test systems use endogenous markers regulated by multiple, often complex regulatory processes, making it difficult to distinguish direct and indirect effects. Moreover, in vitro test systems lack the physiological complexity of EDC metabolism and pharmacokinetics in mammals. Additionally, exposure to environmental EDCs usually involves a mixture of multiple compounds, including in vivo generated metabolites, so the possibility of interactions cannot be ignored. This complexity makes EDC characterization difficult. The Thyroid Hormone Action Indicator (THAI) mouse is a transgenic model that carries a TH-responsive luciferase reporter system, enabling the assessment of tissue-specific TH action. One can evaluate the tissue-specific effects of chemicals on local TH action by quantifying luciferase reporter expression in tissue samples. Furthermore, with in vivo imaging, the THAI mouse model allows for longitudinal studies on the effects of potential EDCs in live animals. This approach provides a powerful tool for testing long-term exposure, complex treatment structures, or withdrawal, as it enables the assessment of changes in local TH action over time in the same animal. This report describes the process of in vivo imaging measurements on THAI mice. The protocol discussed here focuses on developing and imaging hyper- and hypothyroid mice, which can serve as controls. Researchers can adapt or expand the treatments presented to meet their specific needs, offering a foundational approach for further investigation.

Sustained Pituitary T3 Production Explains the T4-mediated TSH Feedback Mechanism.

The regulation of thyroid activity and thyroid hormone (TH) secretion is based on feedback mechanisms that involve the anterior pituitary TSH and medial basal hypothalamus TSH-releasing hormone. Plasma T3 levels can be "sensed" directly by the anterior pituitary and medial basal hypothalamus; plasma T4 levels require local conversion of T4 to T3, which is mediated by the type 2 deiodinase (D2). To study D2-mediated T4 to T3 conversion and T3 production in the anterior pituitary gland, we used mouse pituitary explants incubated with 125I-T4 for 48 hours to measure T3 production at different concentrations of free T4. The results were compared with cultures of D1- or D2-expressing cells, as well as freshly isolated mouse tissue. These studies revealed a unique regulation of the D2 pathway in the anterior pituitary gland, distinct from that observed in nonpituitary tissues. In the anterior pituitary, increasing T4 levels reduced D2 activity slightly but caused a direct increase in T3 production. However, the same changes in T4 levels decreased T3 production in human HSkM cells and murine C2C12 cells (both skeletal muscle) and mouse bone marrow tissue, which reached zero at 50 pM free T4. In contrast, the increase in T4 levels caused the pig kidney LLC-PK1 cells and kidney fragments to proportionally increase T3 production. These findings have important implications for both physiology and clinical practice because they clarify the mechanism by which fluctuations in plasma T4 levels are transduced in the anterior pituitary gland to mediate the TSH feedback mechanism.

Different Hypothalamic Mechanisms Control Decreased Circulating Thyroid Hormone Levels in Infection and Fasting-Induced Non-Thyroidal Illness Syndrome in Male Thyroid Hormone Action Indicator Mice.

Background: Non-Thyroidal Illness Syndrome (NTIS) caused by infection or fasting is hallmarked by reduced circulating thyroid hormone (TH) levels. To better understand the role of local TH-action in the development of NTIS, we assessed tissue-specific changes of TH signaling in Thyroid Hormone Action Indicator (THAI) mice. Methods: NTIS was induced in young adult THAI mice by bacterial lipopolysaccharide (LPS)-administration or by 24 or 48 hours' fasting. Tissue-specific TH-action was assessed by the detection of changes of the Luciferase reporter of THAI mice with quantitative polymerase chain reaction along with tissue-specific examination of regulators of TH metabolism and signaling. Age dependence of revealed alterations of hypothalamic TH-action was also studied in 1-year-old male THAI mice. Results: LPS-treatment increased TH-action in the hypothalamic arcuate nucleus-median eminence (ARC-ME) region preceded by an increase of type 2 deiodinase (D2) expression in the same region and followed by the suppression of proTrh expression in the hypothalamic paraventricular nucleus (PVN). In contrast, LPS decreased both TH-action and D2 activity in the pituitary at both ages. Tshß expression and serum free thyroxine (fT4) and free triiodothyronine (fT3) levels decreased in LPS-treated young adults. Tshß expression and serum fT4 levels were not significantly affected by LPS treatment in aged animals. In contrast to LPS treatment, TH-action remained unchanged in the ARC-ME of 24 and 48 hours fasted animals accompanied with a modest decrease of proTrh expression in the PVN in the 24-hour group. Tshß expression and fT3 level were decreased in both fasted groups, but the fT4 decreased only in the 48 hours fasted animals. Conclusions: Although the hypothalamo-pituitary-thyroid (HPT) axis is inhibited both in LPS and fasting-induced NTIS, LPS achieves this by centrally inducing local hyperthyroidism in the ARC-ME region, while fasting acts without affecting hypothalamic TH signaling. Lack of downregulation of Tshß and fT4 in LPS-treated aged THAI mice suggests age-dependent alterations in the responsiveness of the HPT axis. The LPS-induced tissue-specific hypo-, eu-, and hyperthyroidism in different tissues of the same animal indicate that under certain conditions TH levels alone could be a poor marker of tissue TH signaling. In conclusion, decreased circulating TH levels in these two forms of NTIS are associated with different patterns of hypothalamic TH signaling.

Axonal T3 uptake and transport can trigger thyroid hormone signaling in the brain.

The development of the brain, as well as mood and cognitive functions, are affected by thyroid hormone (TH) signaling. Neurons are the critical cellular target for TH action, with T3 regulating the expression of important neuronal gene sets. However, the steps involved in T3 signaling remain poorly known given that neurons express high levels of type 3 deiodinase (D3), which inactivates both T4 and T3. To investigate this mechanism, we used a compartmentalized microfluid device and identified a novel neuronal pathway of T3 transport and action that involves axonal T3 uptake into clathrin-dependent, endosomal/non-degradative lysosomes (NDLs). NDLs-containing T3 are retrogradely transported via microtubules, delivering T3 to the cell nucleus, and doubling the expression of a T3-responsive reporter gene. The NDLs also contain the monocarboxylate transporter 8 (Mct8) and D3, which transport and inactivate T3, respectively. Notwithstanding, T3 gets away from degradation because D3's active center is in the cytosol. Moreover, we used a unique mouse system to show that T3 implanted in specific brain areas can trigger selective signaling in distant locations, as far as the contralateral hemisphere. These findings provide a pathway for L-T3 to reach neurons and resolve the paradox of T3 signaling in the brain amid high D3 activity.

Cellular and molecular basis of deiodinase-regulated thyroid hormone signaling.

The iodothyronine deiodinases initiate or terminate thyroid hormone action and therefore are critical for the biological effects mediated by thyroid hormone. Over the years, research has focused on their role in preserving serum levels of the biologically active molecule T(3) during iodine deficiency. More recently, a fascinating new role of these enzymes has been unveiled. The activating deiodinase (D2) and the inactivating deiodinase (D3) can locally increase or decrease thyroid hormone signaling in a tissue- and temporal-specific fashion, independent of changes in thyroid hormone serum concentrations. This mechanism is particularly relevant because deiodinase expression can be modulated by a wide variety of endogenous signaling molecules such as sonic hedgehog, nuclear factor-kappaB, growth factors, bile acids, hypoxia-inducible factor-1alpha, as well as a growing number of xenobiotic substances. In light of these findings, it seems clear that deiodinases play a much broader role than once thought, with great ramifications for the control of thyroid hormone signaling during vertebrate development and metamorphosis, as well as injury response, tissue repair, hypothalamic function, and energy homeostasis in adults.

The Hedgehog-inducible ubiquitin ligase subunit WSB-1 modulates thyroid hormone activation and PTHrP secretion in the developing growth plate.

WSB-1 is a SOCS-box-containing WD-40 protein of unknown function that is induced by Hedgehog signalling in embryonic structures during chicken development. Here we show that WSB-1 is part of an E3 ubiquitin ligase for the thyroid-hormone-activating type 2 iodothyronine deiodinase (D2). The WD-40 propeller of WSB-1 recognizes an 18-amino-acid loop in D2 that confers metabolic instability, whereas the SOCS-box domain mediates its interaction with a ubiquitinating catalytic core complex, modelled as Elongin BC-Cul5-Rbx1 (ECS(WSB-1)). In the developing tibial growth plate, Hedgehog-stimulated D2 ubiquitination via ECS(WSB-1) induces parathyroid hormone-related peptide (PTHrP), thereby regulating chondrocyte differentiation. Thus, ECS(WSB-1) mediates a mechanism by which 'systemic' thyroid hormone can effect local control of the Hedgehog-PTHrP negative feedback loop and thus skeletogenesis.

Tanycyte specific ablation of diacylglycerol lipase alpha stimulates the hypothalamic-pituitary-thyroid axis by decreasing the endocannabinoid mediated inhibition of TRH release.

In addition to the hypophysiotropic thyrotropin-releasing hormone (TRH)-synthesizing neurons, a glial cell type, the tanycytes, also play a role in the regulation of the hypothalamic-pituitary-thyroid (HPT) axis. Tanycytes modulate the feedback regulation of the axis by regulating the local thyroid hormone availability in the median eminence where the hypophysiotropic axons terminate. Recently, we showed that tanycytes produce diacylglycerol lipase alpha (DAGLα), the synthesizing enzyme of the endocannabinoid 2-arachidonoylglycerol (2-AG) that inhibits the release of TRH from the hypophysiotropic terminals in median eminence explants. To determine the importance of the endocannabinoid production of tanycytes, adult male Rax-CreERT2//DAGLαfl/fl mice were treated with tamoxifen to induce a tanycyte specific decrease of DAGLα expression (T-DAGLα KO). The effect of this genetic manipulation on the activity of the HPT axis was determined. Tanycyte specific decrease of DAGLα expression resulted in an approximately 2-fold increase of TSHß mRNA level that was accompanied by increased levels of circulating free T4. The TRH mRNA level was, however, not influenced by the genetic manipulation. In addition to the effects on the HPT axis, the T-DAGLα KO mice showed increased fat mass ratio and decreased blood glucose levels. These data indicate that when endocannabinoid release of tanycytes is decreased, the disinhibition of the TRH release induces increased TSH synthesis and higher circulating T4 levels. Thus it suggests that in wild-type mice, tanycytes exert a tonic inhibitory effect on the TRH release of hypophysiotropic axons. Furthermore, the endocannabinoid release of tanycytes also influences glucose homeostasis and fat deposition.

Expression of glucagon-like peptide 1 receptor in neuropeptide Y neurons of the arcuate nucleus in mice.

Glucagon-like peptide 1 (GLP-1) and its agonists exert anorexigenic effect at least partly via acting on GLP-1 receptors (GLP-1R) in the arcuate nucleus (ARC). While the anorexigenic, proopiomelanocortin (POMC) neurons of the ARC were shown previously to express GLP-1R, the putative GLP-1R-content of the orexigenic, neuropeptide Y (NPY) neurons remained so far undetected. As GLP-1R is abundant in the ventromedial ARC, where NPY neurons are located; here, we address the possibility that GLP-1 can act directly on the orexigenic NPY system via GLP-1R. Double-labeling immunocytochemistry and in situ hybridization were performed on tissues of adult male mice to detect GLP-1R in NPY neurons. In double-immunolabeled preparations, GLP-1R-immunoreactivity was observed in NPY neurons and in axons ensheathing the majority of NPY neurons. Ultrastructural studies confirmed that GLP-1R-immunoreactivity is associated with the outer membrane of NPY perikarya as well as with axons forming symmetric type, inhibitory synapses on NPY-containing neurons. Double-labeling in situ hybridization experiments demonstrated the expression of GLP-1R mRNA in approximately 20% of NPY mRNA-containing neurons of the ARC. In summary, our data demonstrate the presence of GLP-1R protein and mRNA in NPY neurons of ARC and also reveal the innervation of NPY neurons by GLP-1R-containing inhibitory neurons. These observations suggest that GLP-1 signaling can influence NPY neurons both directly and indirectly. Furthermore, GLP-1 signaling on energy homeostasis appears to involve both direct and indirect effects of GLP-1 on the orexigenic NPY neurons, in addition to the previously known effects via the anorexigenic POMC neuronal system.

Origin of thyrotropin-releasing hormone neurons that innervate the tuberomammillary nuclei.

Hypophysiotropic thyrotropin-releasing hormone (TRH) neurons function as metabolic sensors that regulate the thyroid axis and energy homeostasis. Less is known about the role of other hypothalamic TRH neurons. As central administration of TRH decreases food intake and increases histamine in the tuberomammillary nuclei (TMN), and TMN histamine neurons are densely innervated by TRH fibers from an unknown origin, we mapped the location of TRH neurons that project to the TMN. The retrograde tracer, cholera toxin B subunit (CTB), was injected into the TMN E1-E2, E4-E5 subdivisions of adult Sprague-Dawley male rats. TMN projecting neurons were observed in the septum, preoptic area, bed nucleus of the stria terminalis (BNST), perifornical area, anterior paraventricular nucleus, peduncular and tuberal lateral hypothalamus (TuLH), suprachiasmatic nucleus and medial amygdala. However, CTB/pro-TRH178-199 double-labeled cells were only found in the TuLH. The specificity of the retrograde tract-tracing result was confirmed by administering the anterograde tracer, Phaseolus vulgaris leuco-agglutinin (PHAL) into the TuLH. Double-labeled PHAL-pro-TRH boutons were identified in all subdivisions of the TMN. TMN neurons double-labeled for histidine decarboxylase (Hdc)/PHAL, Hdc/Trh receptor (Trhr), and Hdc/Trh. Further confirmation of a TuLH-TRH neuronal projection to the TMN was established in a transgenic mouse that expresses Cre recombinase in TRH-producing cells following microinjection of a Cre recombinase-dependent AAV that expresses mCherry into the TuLH. We conclude that, in rodents, the TRH innervation of TMN originates in part from TRH neurons in the TuLH, and that this TRH population may contribute to regulate energy homeostasis through histamine Trhr-positive neurons of the TMN.

Triiodothyronine (T3) promotes brown fat hyperplasia via thyroid hormone receptor α mediated adipocyte progenitor cell proliferation.

The thyroid hormone (TH)-controlled recruitment process of brown adipose tissue (BAT) is not fully understood. Here, we show that long-term treatment of T3, the active form of TH, increases the recruitment of thermogenic capacity in interscapular BAT of male mice through hyperplasia by promoting the TH receptor α-mediated adipocyte progenitor cell proliferation. Our single-cell analysis reveals the heterogeneous nature and hierarchical trajectory within adipocyte progenitor cells of interscapular BAT. Further analyses suggest that T3 facilitates cell state transition from a more stem-like state towards a more committed adipogenic state and promotes cell cycle progression towards a mitotic state in adipocyte progenitor cells, through mechanisms involving the action of Myc on glycolysis. Our findings elucidate the mechanisms underlying the TH action in adipocyte progenitors residing in BAT and provide a framework for better understanding of the TH effects on hyperplastic growth and adaptive thermogenesis in BAT depot at a single-cell level.