A Phase 3, Randomized, Controlled Trial of Resmetirom in NASH with Liver Fibrosis.

BACKGROUND: Nonalcoholic steatohepatitis (NASH) is a progressive liver disease with no approved treatment. Resmetirom is an oral, liver-directed, thyroid hormone receptor beta-selective agonist in development for the treatment of NASH with liver fibrosis. METHODS: We are conducting an ongoing phase 3 trial involving adults with biopsy-confirmed NASH and a fibrosis stage of F1B, F2, or F3 (stages range from F0 [no fibrosis] to F4 [cirrhosis]). Patients were randomly assigned in a 1:1:1 ratio to receive once-daily resmetirom at a dose of 80 mg or 100 mg or placebo. The two primary end points at week 52 were NASH resolution (including a reduction in the nonalcoholic fatty liver disease [NAFLD] activity score by ≥2 points; scores range from 0 to 8, with higher scores indicating more severe disease) with no worsening of fibrosis, and an improvement (reduction) in fibrosis by at least one stage with no worsening of the NAFLD activity score. RESULTS: Overall, 966 patients formed the primary analysis population (322 in the 80-mg resmetirom group, 323 in the 100-mg resmetirom group, and 321 in the placebo group). NASH resolution with no worsening of fibrosis was achieved in 25.9% of the patients in the 80-mg resmetirom group and 29.9% of those in the 100-mg resmetirom group, as compared with 9.7% of those in the placebo group (P<0.001 for both comparisons with placebo). Fibrosis improvement by at least one stage with no worsening of the NAFLD activity score was achieved in 24.2% of the patients in the 80-mg resmetirom group and 25.9% of those in the 100-mg resmetirom group, as compared with 14.2% of those in the placebo group (P<0.001 for both comparisons with placebo). The change in low-density lipoprotein cholesterol levels from baseline to week 24 was -13.6% in the 80-mg resmetirom group and -16.3% in the 100-mg resmetirom group, as compared with 0.1% in the placebo group (P<0.001 for both comparisons with placebo). Diarrhea and nausea were more frequent with resmetirom than with placebo. The incidence of serious adverse events was similar across trial groups: 10.9% in the 80-mg resmetirom group, 12.7% in the 100-mg resmetirom group, and 11.5% in the placebo group. CONCLUSIONS: Both the 80-mg dose and the 100-mg dose of resmetirom were superior to placebo with respect to NASH resolution and improvement in liver fibrosis by at least one stage. (Funded by Madrigal Pharmaceuticals; MAESTRO-NASH ClinicalTrials.gov number, NCT03900429.).

Syringic acid, a novel thyroid hormone receptor-ß agonist, ameliorates propylthiouracil-induced thyroid toxicity in rats.

The aim of this study was to evaluate the potential of syringic acid (SA) against propylthiouracil (PTU)-induced hypothyroidism in rats. SA at a prestandardized dose, 50 mg/kg/day, was orally administered to PTU-induced hypothyroid rats for 30 days, and alterations in the levels of serum triiodothyronine (T3 ), thyroxine (T4 ), thyrotropin (TSH), alanine transaminase (ALT), and aspartate transaminase (AST); tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6); total cholesterol (CHOL) and triglycerides (TG); hepatic lipid peroxidation (LPO) and antioxidants (superoxide dismutase, catalase, glutathione peroxidase, and glutathione content), as well as histological changes in liver and thyroid were examined. The molecular interactions of the ligand, SA, with thyroid-related protein targets, such as human thyroid hormone receptor ß (hTRß), and thyroid peroxidase (TPO) protein, were studied using molecular docking. Whereas in hypothyroid animals, T4 , T3 , and antioxidants were decreased, there was an increase in TSH, TNF-α, IL-6, ALT, AST, and hepatic LPO; administration of SA in PTU-induced animals reversed all these indices to near normal levels. SA also improved the histological features of liver and thyroid gland. Our study clearly demonstrates SA as a novel thyroid agonist for augmenting the thyroid functions in rats. Molecular docking analysis reveals that SA possesses good binding affinity toward both the targets, hTRß and TPO. Through this approach, for the first time we provide the evidence for SA as a novel thyroid agonist and suggest a receptor-mediated mechanism for its thyroid stimulatory potential.

Effects of Resmetirom on Noninvasive Endpoints in a 36-Week Phase 2 Active Treatment Extension Study in Patients With NASH.

Resmetirom (MGL-3196), a selective thyroid hormone receptor-ß agonist, was evaluated in a 36-week paired liver biopsy study (NCT02912260) in adults with biopsy-confirmed nonalcoholic steatohepatitis (NASH). The primary endpoint was relative liver fat reduction as assessed by MRI-proton density fat fraction (MRI-PDFF), and secondary endpoints included histopathology. Subsequently, a 36-week active treatment open-label extension (OLE) study was conducted in 31 consenting patients (including 14 former placebo patients) with persistently mild to markedly elevated liver enzymes at the end of the main study. In patients treated with resmetirom (80 or 100 mg orally per day), MRI-PDFF reduction at OLE week 36 was -11.1% (1.5%) mean reduction (standard error [SE]; P < 0.0001) and -52.3% (4.4%) mean relative reduction, P < 0.0001. Low-density lipoprotein (LDL) cholesterol (-26.1% [4.5%], P < 0.0001), apolipoprotein B (-23.8% [3.0%], P < 0.0001), and triglycerides (-19.6% [5.4%], P = 0.0012; -46.1 [14.5] mg/dL, P = 0.0031) were reduced from baseline. Markers of fibrosis were reduced, including liver stiffness assessed by transient elastography (-2.1 [0.8] mean kilopascals [SE], P = 0.015) and N-terminal type III collagen pro-peptide (PRO-C3) (-9.8 [2.3] ng/mL, P = 0.0004 (baseline ≥ 10 ng/mL). In the main and OLE studies, PRO-C3/C3M (matrix metalloproteinase-degraded C3), a marker of net fibrosis formation, was reduced in resmetirom-treated patients (-0.76 [-1.27, -0.24], P = 0.0044 and -0.68, P < 0.0001, respectively). Resmetirom was well tolerated, with few, nonserious adverse events. Conclusion: The results of this 36-week OLE study support the efficacy and safety of resmetirom at daily doses of 80 mg and 100 mg, used in the ongoing phase 3 NASH study, MAESTRO-NASH (NCT03900429). The OLE study demonstrates a potential for noninvasive assessments to monitor the response to resmetirom from an individual patient with NASH.

Regulation of gene transcription by thyroid hormone receptor ß agonists in clinical development for the treatment of non-alcoholic steatohepatitis (NASH).

Thyroid hormones are important modulators of metabolic activity in mammals and alter cholesterol and fatty acid levels through activation of the nuclear thyroid hormone receptor (THR). Currently, there are several THRß agonists in clinical trials for the treatment of non-alcoholic steatohepatitis (NASH) that have demonstrated the potential to reduce liver fat and restore liver function. In this study, we tested three THRß-agonism-based NASH treatment candidates, GC-1 (sobetirome), MGL-3196 (resmetirom), and VK2809, and compared their selectivity for THRß and their ability to modulate the expression of genes specific to cholesterol and fatty acid biosynthesis and metabolism in vitro using human hepatic cells and in vivo using a rat model. Treatment with GC-1 upregulated the transcription of CPT1A in the human hepatocyte-derived Huh-7 cell line with a dose-response comparable to that of the native THR ligand, triiodothyronine (T3). VK2809A (active parent of VK2809), MGL-3196, and VK2809 were approximately 30-fold, 1,000-fold, and 2,000-fold less potent than T3, respectively. Additionally, these relative potencies were confirmed by quantification of other direct gene targets of THR, namely, ANGPTL4 and DIO1. In primary human hepatocytes, potencies were conserved for every compound except for VK2809, which showed significantly increased potency that was comparable to that of its active counterpart, VK2809A. In high-fat diet fed rats, a single dose of T3 significantly reduced total cholesterol levels and concurrently increased liver Dio1 and Me1 RNA expression. MGL-3196 treatment resulted in concentration-dependent decreases in total and low-density lipoprotein cholesterol with corresponding increases in liver gene expression, but the compound was significantly less potent than T3. In conclusion, we have implemented a strategy to rank the efficacy of THRß agonists by quantifying changes in the transcription of genes that lead to metabolic alterations, an effect that is directly downstream of THR binding and activation.

Design, synthesis and biological evaluation of novel TRß selective agonists sustained by ADME-toxicity analysis.

Although triiodothyronine (T3) induces several beneficial effects on lipid metabolism, its use is hampered by toxic side-effects, such as tachycardia, arrhythmia, heart failure, bone and muscle catabolism and mood disturbances. Since the α isoform of thyroid hormone receptors (TRs) is the main cause of T3-related harmful effects, several efforts have been made to develop selective agonists of the ß isoform that could induce some beneficial effects (i.e. lowering triglyceride and cholesterol levels reducing obesity and improving metabolic syndrome), while overcoming most of the adverse T3-dependent side effects. Herein, we describe the drug discovery process sustained by ADME-Toxicity analysis that led us to identify novel agonists with selectivity for the isoform TRß and an acceptable off-target and absorption, distribution metabolism, excretion and toxicity (ADME-Tox) profile. Within the small series of compounds synthesized, derivatives 1 and 3, emerge from this analysis as "potentially safe" to be engaged in preclinical studies. In in vitro investigation proved that both compounds were able to reduce lipid accumulation in HepG2 and promote lipolysis with comparable effects to those elicited by T3, used as reference drug. Moreover, a preliminary in vivo study confirmed the apparent lack of toxicity, thus suggesting compounds 1 and 3 as new potential TRß-selective thyromimetics.

Resmetirom (MGL-3196) for the treatment of non-alcoholic steatohepatitis: a multicentre, randomised, double-blind, placebo-controlled, phase 2 trial.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) is characterised by hepatic steatosis, inflammation, hepatocellular injury, and progressive liver fibrosis. Resmetirom (MGL-3196) is a liver-directed, orally active, selective thyroid hormone receptor-ß agonist designed to improve NASH by increasing hepatic fat metabolism and reducing lipotoxicity. We aimed to assess the safety and efficacy of resmetirom in patients with NASH. METHODS: MGL-3196-05 was a 36-week randomised, double-blind, placebo-controlled study at 25 centres in the USA. Adults with biopsy confirmed NASH (fibrosis stages 1-3) and hepatic fat fraction of at least 10% at baseline when assessed by MRI-proton density fat fraction (MRI-PDFF) were eligible. Patients were randomly assigned 2:1 by a computer-based system to receive resmetirom 80 mg or matching placebo, orally once a day. Serial hepatic fat measurements were obtained at weeks 12 and 36, and a second liver biopsy was obtained at week 36. The primary endpoint was relative change in MRI-PDFF assessed hepatic fat compared with placebo at week 12 in patients who had both a baseline and week 12 MRI-PDFF. This trial is registered with ClinicalTrials.gov, number NCT02912260. FINDINGS: 348 patients were screened and 84 were randomly assigned to resmetirom and 41 to placebo at 18 sites in the USA. Resmetirom-treated patients (n=78) showed a relative reduction of hepatic fat compared with placebo (n=38) at week 12 (-32·9% resmetirom vs -10·4% placebo; least squares mean difference -22·5%, 95% CI -32·9 to -12·2; p<0·0001) and week 36 (-37·3% resmetirom [n=74] vs -8·5 placebo [n=34]; -28·8%, -42·0 to -15·7; p<0·0001). Adverse events were mostly mild or moderate and were balanced between groups, except for a higher incidence of transient mild diarrhoea and nausea with resmetirom. INTERPRETATION: Resmetirom treatment resulted in significant reduction in hepatic fat after 12 weeks and 36 weeks of treatment in patients with NASH. Further studies of resmetirom will allow assessment of safety and effectiveness of resmetirom in a larger number of patients with NASH with the possibility of documenting associations between histological effects and changes in non-invasive markers and imaging. FUNDING: Madrigal Pharmaceuticals.

A Liver-Specific Thyromimetic, VK2809, Decreases Hepatosteatosis in Glycogen Storage Disease Type Ia.

Background: Glycogen storage disease type Ia (GSD Ia), also known as von Gierke disease, is the most common glycogen storage disorder. It is caused by the deficiency of glucose-6-phosphatase, the enzyme that catalyzes the final step of gluconeogenesis and glycogenolysis. The accumulation of glucose-6-phosphate leads to increased glycogen and triglyceride levels in the liver. Patients with GSD Ia can develop steatohepatitis, cirrhosis, and increased risk for hepatocellular adenomas and carcinomas. We previously showed that animal models of GSD Ia had defective autophagy and dysfunctional mitochondria. In this study, we examined the effect of VK2809, a liver-specific thyroid hormone receptor ß agonist, on hepatic steatosis, autophagy, and mitochondrial biogenesis in a mouse model of GSD Ia. Methods:G6pc-/--deficient (GSD Ia) mice were treated with VK2809 or vehicle control by daily intraperitoneal injection for four days. The hepatic triglyceride and glycogen were determined by biochemical assays. Autophagy and mitochondrial biogenesis were measured by Western blotting for key autophagy and mitochondrial markers. Results: VK2809 treatment decreased hepatic mass and triglyceride content in GSD Ia mice. VK2809 stimulated hepatic autophagic flux as evidenced by increased microtubule-associated protein light chain 3-II (LC3B-II), decreased p62 protein levels, activation of AMP-activated protein kinase (AMPK), inhibition of the mammalian target of rapamycin (mTOR) signaling, enhancement of protein levels of ATG5-ATG12, and increased lysosomal protein expression. VK2809 also increased the expression of carnitine palmitoyltransferase 1a (CPT1α) and fibroblast growth factor 21 (FGF21), as well as mitochondrial biogenesis to promote mitochondrial ß-oxidation. Conclusions: In summary, VK2809 treatment decreased hepatic triglyceride levels in GSD Ia mice through its simultaneous restoration of autophagy, mitochondrial biogenesis, and ß-oxidation of fatty acids. Liver-specific thyromimetics represent a potential therapy for hepatosteatosis in GSD Ia as well as nonalcoholic fatty liver disease.

GC-1: A Thyromimetic With Multiple Therapeutic Applications in Liver Disease.

Thyroid hormones (THs), namely, 3,5,3'-triiodo-l-thyronine (T3) and 3,5,3',5'-tetraiodo-l-thyronine (thyroxine or T4), influence a variety of physiological processes that have important implications in fetal development, metabolism, cell growth, and proliferation. While THs elicit several beneficial effects on lipid metabolism and improve myocardial contractility, these therapeutically desirable effects are associated to a thyrotoxic state that severely limits the possible use of THs as therapeutic agents. Therefore, several efforts have been made to develop T3 analogs that could retain the beneficial actions (triglyceride, cholesterol, obesity, and body mass lowering) without the adverse TH-dependent side effects. This goal was achieved by the synthesis of TRß-selective agonists. In this review, we summarize the current knowledge on the effects of one of the best characterized TH analogs, the TRß1-selective thyromimetic, GC-1. In particular, we review some of the effects of GC-1 on different liver disorders, with reference to its possible clinical application. A brief comment on the possible therapeutic use of GC-1 in extrahepatic disorders is also included.

The identification of the metabolites of chlorothalonil in zebrafish (Danio rerio) and their embryo toxicity and endocrine effects at environmentally relevant levels.

Chlorothalonil is a broad spectrum fungicide with high annual production and environmental contamination. Despite its high consumption, studies regarding the potential ecological risks of chlorothalonil, especially its metabolites, to aquatic organisms are still limited. In this study, we selected the zebrafish (Danio rerio) as the in vivo model and first identified the metabolite (4-hydroxychlorothalonil) of chlorothalonil in zebrafish by tandem quadrupole/orthogonal-acceleration time-of-flight (Q-TOF). Then, the in vivo and in vitro models were applied to comprehensively estimate the embryo toxicity and potential endocrine effect of chlorothalonil and 4-hydroxychlorothalonil. The data from zebrafish embryo toxicity showed that the lowest observed effect concentrations of both chlorothalonil and 4-hydroxychlorothalonil were 50 µg/L after 96 h of exposure. The mortality rate of the 4-hydroxychlorothalonil was 2.6-fold higher than that of the parent compound at the concentration of 50 µg/L. Dual-luciferase reporter gene assays indicated that both chlorothalonil and 4-hydroxychlorothalonil exerted estrogen receptor α (ERα) agonist activity with REC20 values of 2.4 × 10-8 M and 3.6 × 10-8 M, respectively. However, only 4-hydroxychlorothalonil exhibited both thyroid receptor ß (TRß) agonistic and antagonistic activities. Lastly, we employed molecular docking to predict the binding affinity of chlorothalonil and 4-hydroxychlorothalonil with ERα or TRß. The results revealed that the potential endocrine effect of chlorothalonil and 4-hydroxychlorothaloni might be attributed to the different binding affinities with the receptors. In conclusion, our studies revealed that 4-hydroxychlorothalonil exhibited potent endocrine-disrupting effects compared to its parent compound, chlorothalonil. The results provided here remind us that the assessment of the potential ecological and health risks of the metabolites of fungicides in addition to their parent compounds should arouse great concerns.

Thyroid Hormone Receptor ß Agonist Induces ß-Catenin-Dependent Hepatocyte Proliferation in Mice: Implications in Hepatic Regeneration.

Triiodothyronine (T3) induces hepatocyte proliferation in rodents. Recent work has shown molecular mechanism for T3's mitogenic effect to be through activation of ß-catenin signaling. Since systemic side effects of T3 may preclude its clinical use, and hepatocytes mostly express T3 hormone receptor ß (TRß), we investigated if selective TRß agonists like GC-1 may also have ß-catenin-dependent hepatocyte mitogenic effects. Here we studied the effect of GC-1 and T3 in conditional knockouts of various Wnt pathway components. We also assessed any regenerative advantage of T3 or GC-1 when given prior to partial hepatectomy in mice. Mice administered GC-1 showed increased pSer675-ß-catenin, cyclin D1, BrdU incorporation, and PCNA. No abnormalities in liver function tests were noted. GC-1-injected liver-specific ß-catenin knockouts (ß-catenin LKO) showed decreased proliferation when compared to wild-type littermates. To address if Wnt signaling was required for T3- or GC-1-mediated hepatocyte proliferation, we used LRP5-6-LKO, which lacks the two redundant Wnt coreceptors. Surprisingly, decreased hepatocyte proliferation was also evident in LRP5-6-LKO in response to T3 and GC-1, despite increased pSer675-ß-catenin. Further, increased levels of active ß-catenin (hypophosphorylated at Ser33, Ser37, and Thr41) were evident after T3 and GC-1 treatment. Finally, mice pretreated with T3 or GC-1 for 7 days followed by partial hepatectomy showed a significant increase in hepatocyte proliferation both at the time (T0) and 24 h after surgery. In conclusion, like T3, TRß-selective agonists induce hepatocyte proliferation through ß-catenin activation via both PKA- and Wnt-dependent mechanisms and confer a regenerative advantage following surgical resection. Hence, these agents may be useful regenerative therapies in liver transplantation or other surgical settings.

Thyroid hormone receptor-ß1 signaling is critically involved in regulating secondary ossification via promoting transcription of the Ihh gene in the epiphysis.

Thyroid hormone (TH) action is mediated through two nuclear TH receptors, THRα and THRß. Although the role of THRα is well established in bone, less is known about the relevance of THRß-mediated signaling in bone development. On ther basis of our recent finding that TH signaling is essential for initiation and formation of secondary ossification center, we evaluated the role of THRs in mediating TH effects on epiphysial bone formation. Two-day treatment of TH-deficient Tshr(-/-) mice with TH increased THRß1 mRNA level 3.4-fold at day 7 but had no effect on THRα1 mRNA level at the proximal tibia epiphysis. Treatment of serum-free cultures of tibias from 3-day-old mice with T3 increased THRß1 expression 2.1- and 13-fold, respectively, at 24 and 72 h. Ten-day treatment of Tshr(-/-) newborns (days 5-14) with THRß1 agonist GC1 at 0.2 or 2.0 µg/day increased BV/TV at day 21 by 225 and 263%, respectively, compared with vehicle treatment. Two-day treatment with GC1 (0.2 µg/day) increased expression levels of Indian hedgehog (Ihh) 100-fold, osterix 15-fold, and osteocalcin 59-fold compared with vehicle at day 7 in the proximal tibia epiphysis. Gel mobility shift assay demonstrated that a putative TH response element in the distal promoter of mouse Ihh gene interacted with THRß1. GC1 treatment (1 nM) increased Ihh distal promoter activity 20-fold after 48 h in chondroctyes. Our data suggest a novel role for THRß1 in secondary ossification at the epiphysis that involves transcriptional upregulation of Ihh gene.

Thyroid Hormone Receptor α Plays an Essential Role in Male Skeletal Muscle Myoblast Proliferation, Differentiation, and Response to Injury.

Thyroid hormone plays an essential role in myogenesis, the process required for skeletal muscle development and repair, although the mechanisms have not been established. Skeletal muscle develops from the fusion of precursor myoblasts into myofibers. We have used the C2C12 skeletal muscle myoblast cell line, primary myoblasts, and mouse models of resistance to thyroid hormone (RTH) α and ß, to determine the role of thyroid hormone in the regulation of myoblast differentiation. T3, which activates thyroid hormone receptor (TR) α and ß, increased myoblast differentiation whereas GC1, a selective TRß agonist, was minimally effective. Genetic approaches confirmed that TRα plays an important role in normal myoblast proliferation and differentiation and acts through the Wnt/ß-catenin signaling pathway. Myoblasts with TRα knockdown, or derived from RTH-TRα PV (a frame-shift mutation) mice, displayed reduced proliferation and myogenic differentiation. Moreover, skeletal muscle from the TRα1PV mutant mouse had impaired in vivo regeneration after injury. RTH-TRß PV mutant mouse model skeletal muscle and derived primary myoblasts did not have altered proliferation, myogenic differentiation, or response to injury when compared with control. In conclusion, TRα plays an essential role in myoblast homeostasis and provides a potential therapeutic target to enhance skeletal muscle regeneration.

The thyroid hormone receptor β-selective agonist GC-1 does not affect tolerance to exercise in hypothyroid rats

Objective Investigate the effect of GC-1 on tolerance to exercise in rats with experimental hypothyroidism. Materials and methods Hypothyroidism was induced with methimazole sodium and perchlorate treatment. Six groups with eight animals were studied: control group (C), hypothyroid group without treatment (HYPO); hypothyroidism treated with physiological doses of tetraiodothyronine (T4) or 10 times higher (10×T4); hypothyroidism treated with equal molar doses of GC-1 (GC-1) or 10 times higher (10×GC-1). After eight weeks, each animal underwent an exercise tolerance test by measuring the time (seconds), in which the rats were swimming with a load attached to their tails without being submerging for more than 10 sec. After the test, the animals were killed, and blood samples were collected for biochemical analysis, and the heart and soleus muscle were removed for weighing and morphometric analysis of the cardiomyocyte. Results Hypothyroidism significantly reduced tolerance to exercise and, treatment with GC-1 1× or T4 in physiological doses recover tolerance test to normal parameters. However, high doses of T4 also decreased tolerance to physical exercise. Conversely, ten times higher doses of GC-1 did not impair tolerance to exercise. Interestingly, hypothyroidism, treated or not with T4 in a physiological range, GC-1 or even high doses of GC-1 (10X) did not change cardiomyocyte diameters and relative weight of the soleus muscle. In contrast, higher doses of T4 significantly increased cardiomyocyte diameter and induced atrophy of the soleus muscle. Conclusion Unlike T4, GC-1 in high doses did not modify tolerance to physical exercise in the rats with hypothyroidism. .

Peripheral targets in obesity treatment: a comprehensive update.

Obesity is a major epidemic of our time and is associated with diseases such as metabolic syndrome, type 2 diabetes mellitus and atherosclerotic cardiovascular disease. Although weight loss drugs, when accompanied by diet and exercise, could be a very helpful medical tool in treating obese or overweight patients, their usefulness has been questioned due to the complexity of this type of medication, which regards a plethora of issues such as efficacy and safety of the drug and also risks and benefits among different patients. In general, obesity drugs that target peripheral pathophysiological mechanisms can be divided into two main categories. The first category includes anti-obesity agents able to reduce or limit energy absorption, such as pancreatic lipase and microsomal triglyceride transfer protein inhibitors. The second category consists of a heterogeneous group of compounds aiming to decrease fat mass by increasing energy expenditure or by redistributing adipose tissue. Angiogenesis inhibitors, beta-3 receptor agonists, sirtuin-I activators, diazoxide and other molecules belong to this group. The glucagon-like peptide-1 receptor agonists consist the third category of peripheral anti-obesity agents discussed therein. This review aims to provide a general overview of the molecules and substances that are already or could potentially be used as peripheral anti-obesity drugs, the molecular mechanisms by which they act, as well as their current stage of development, production and/or availability.

The ability of thyroid hormone receptors to sense t4 as an agonist depends on receptor isoform and on cellular cofactors.

T4 (3,5,3',5'-tetraiodo-l-thyronine) is classically viewed as a prohormone that must be converted to the T3 (3,5,3'-triiodo-l-thyronine) form for biological activity. We first determined that the ability of reporter genes to respond to T4 and to T3 differed for the different thyroid hormone receptor (TR) isoforms, with TRα1 generally more responsive to T4 than was TRß1. The response to T4 vs T3 also differed dramatically in different cell types in a manner that could not be attributed to differences in deiodinase activity or in hormone affinity, leading us to examine the role of TR coregulators in this phenomenon. Unexpectedly, several coactivators, such as steroid receptor coactivator-1 (SRC1) and thyroid hormone receptor-associated protein 220 (TRAP220), were recruited to TRα1 nearly equally by T4 as by T3 in vitro, indicating that TRα1 possesses an innate potential to respond efficiently to T4 as an agonist. In contrast, release of corepressors, such as the nuclear receptor coreceptor NCoRω, from TRα1 by T4 was relatively inefficient, requiring considerably higher concentrations of this ligand than did coactivator recruitment. Our results suggest that cells, by altering the repertoire and abundance of corepressors and coactivators expressed, may regulate their ability to respond to T4, raising the possibility that T4 may function directly as a hormone in specific cellular or physiological contexts.

Thyroid hormone status regulates the expression of secretory phospholipases.

Thyroid hormone (T3) stimulates various metabolic pathways and the hepatic actions of T3 are mediated primarily through the thyroid hormone receptor beta (TRß). Hypothyroidism has been linked with low grade inflammation, elevated risk of hepatic steatosis and atherosclerosis. Secretory phospholipases (sPLA2) are associated with inflammation, hyperlipidemia and atherosclerosis. Due to potential linkage between thyroid hormone and sPLA2, we investigated the effect of thyroid hormone status on the regulation of secretory phospholipases in mice, rats and human liver. T3 suppressed the expression of the sPLA2 group IIa (PLA2g2a) gene in the liver of BALB/c mice and C57BL/6 transgenic mice expressing the human PLA2g2a. PLA2g2a was elevated with hypothyroidism and high fat diets which may contribute to the low grade inflammation associated with hypothyroidism and diet induced obesity. We also examined the effects of the TRß agonist eprotirome on hepatic gene regulation. We observed that eprotirome inhibited the expression of selected sPLA2 genes and furthermore the cytokine mediated induction PLA2g2a was suppressed. In addition, eprotirome induced genes involved in fatty acid oxidation and cholesterol clearance while inhibiting lipogenic genes. Our results indicate that in vivo thyroid hormone status regulates the abundance of sPLA2 and the inhibition of PLA2g2a by T3 is conserved across species. By regulating sPLA2 genes, T3 may impact processes associated with atherosclerosis and inflammation and TRß agonists may ameliorate inflammation and hyperlipidemia.

3,5-T2 is an alternative ligand for the thyroid hormone receptor ß1.

Several liganded nuclear receptors have alternative ligands acting in a tissue-specific fashion and playing important biological roles. We present evidence that 3,5-diiodothyronine (T(2)), a naturally occurring iodothyronine that results from T(3) outer-ring deiodination, is an alternative ligand for thyroid hormone receptor ß1 (TRß1). In tilapia, 2 TRß isoforms differing by 9 amino acids in the ligand-binding domain were cloned. Binding and transactivation studies showed that T(2) activates the human and the long tilapia TRß1 isoform, but not the short one. A chimeric human TRß1 (hTRß1) that contained the 9-amino-acid insert showed no response to T(2), suggesting that the conformation of the hTRß1 naturally allows T(2) binding and that other regions of the receptor are implicated in TR activation by T(2). Indeed, further analysis showed that the N terminus is essential for T(2)-mediated transactivation but not for that by T(3) in the long and hTRß1, suggesting a functional interaction between the N-terminal domain and the insertion in the ligand-binding domain. To establish the functional relevance of T(2)-mediated TRß1 binding and activation, mRNA expression and its regulation by T(2) and T(3) was evaluated for both isoforms. Our data show that long TRß1expression is 10(6)-fold higher than that of the short isoform, and T(3) and T(2) differentially regulate the expression of these 2 TRß1 isoforms in vivo. Taken together, our results prompted a reevaluation of the role and mechanism of action of thyroid hormone metabolites previously believed to be inactive. More generally, we propose that classical liganded receptors are only partially locked to very specific ligands and that alternative ligands may play a role in the tissue-specific action of receptors.

Thyroid hormone receptor and liver X receptor competitively up-regulate human selective Alzheimer's disease indicator-1 gene expression at the transcriptional levels.

Selective Alzheimer's disease (AD) indicator-1 (Seladin-1) gene has been identified as a gene, whose expression is down-regulated in the vulnerable region in the brain of AD patients. Thyroid hormone (TH) is important to maintain the function of central nervous system and TH receptor (TR) is known to crosstalk with liver X receptor (LXR) on the lipid metabolism-related gene promoter. Recently, we have demonstrated that TR-ß up-regulates the mouse Seladin-1 gene promoter at the transcriptional levels and LXR-α compensates the promoter activation only when the thyroid function is insufficient. In the current study, we have identified that TH and an LXR artificial agonist, TO901317 (TO) activated the human Seladin-1 promoter (-1024/+57 base pair (bp)) including consensus TH response element (TRE) half site (site A: -381 to -375 bp), and the site A mutation deteriorated the activation by TH and TO. Both TR-ß and LXR-α heterodimerize with retinoid X receptor (RXR)-α on the site A, and chromatin immunoprecipitation (ChIP) assay revealed that TR-ß, LXR-α and RXR-α are recruited to the site A. Moreover, TR-ß and LXR-α functionally compete for the promoter activation in CV1 cells. Taken together, we concluded that TR-ß and LXR-α competitively up-regulate the human Seladin-1 promoter, sharing the same response element, site A.

Cardiac specific effects of thyroid hormone analogues.

There is significant interest in development of thyroid hormone analogues to harness specific properties as therapeutic agents for a variety of clinical indications including obesity, hypercholesterolemia, heart failure, and thyrotoxicosis. To date, most analogues have been designed to target liver specific effects, which can promote weight loss and lipid lowering through either tissue specific uptake or thyroid hormone receptor (TR) ß isoform selectivity at the same time minimizing the unwanted cardiac and bone effects. We have developed a molecular biomarker assay to study the induction of the transcription of the cardiac specific α-myosin heavy chain (MHC) gene as a more sensitive and specific measure of thyroid hormone action on cardiac myocytes. We tested 5 TRß and 1 TRα selective agonists as well as 2 putative TR antagonists in our α-MHC hnRNA assay. Using reverse transcription and polymerase chain reaction, we measured the induction of the α-MHC primary transcript in response to administration of drug. The TRα and only 2 of the TRß agonists were highly active, when compared to the effect of T3, at the level of the cardiac myocyte. In addition, our data suggests that the reason that the antagonist NH-3 is not able to block the T3-mediated induction of α-MHC is that it does not get transported into the cardiac myocyte. Our data suggest that this assay will be useful in preclinical studies of the potential cardiac specific effects of thyroid hormone analogues and that predictions of function based on structure are not necessarily accurate or complete.

In vivo activity of the thyroid hormone receptor beta- and α-selective agonists GC-24 and CO23 on rat liver, heart, and brain.

Thyroid hormone analogs with selective actions through specific thyroid hormone receptor (TR) subtypes are of great interest. They might offer the possibility of mimicking physiological actions of thyroid hormone with receptor subtype or tissue specificity with therapeutic aims. They are also pharmacological tools to dissect biochemical pathways mediated by specific receptor subtypes, in a complementary way to mouse genetic modifications. In this work, we studied the in vivo activity in developing rats of two thyroid hormone agonists, the TRß-selective GC-24 and the TRα-selective CO23. Our principal goal was to check whether these compounds were active in the rat brain. Analog activity was assessed by measuring the expression of thyroid hormone target genes in liver, heart, and brain, after administration to hypothyroid rats. GC-24 was very selective for TRß and lacked activity on the brain. On the other hand, CO23 was active in liver, heart, and brain on genes regulated by either TRα or TRß. This compound, previously shown to be TRα-selective in tadpoles, displayed no selectivity in the rat in vivo.