For more than a century, thyroid hormones (THs) have been known to exert powerful catabolic effects, leading to weight loss. Although much has been learned about the molecular mechanisms used by TH receptors (TRs) to regulate gene expression, little is known about the mechanisms by which THs increase oxidative metabolism. Here, we report that TH stimulation of fatty acid β-oxidation is coupled with induction of hepatic autophagy to deliver fatty acids to mitochondria in cell culture and in vivo. Furthermore, blockade of autophagy by autophagy-related 5 (ATG5) siRNA markedly decreased TH-mediated fatty acid β-oxidation in cell culture and in vivo. Consistent with this model, autophagy was altered in livers of mice expressing a mutant TR that causes resistance to the actions of TH as well as in mice with mutant nuclear receptor corepressor (NCoR). These results demonstrate that THs can regulate lipid homeostasis via autophagy and help to explain how THs increase oxidative metabolism. IntroductionThyroid hormones (THs) have been known to stimulate basal metabolic rate for over a century (1, 2). Subsequent studies showed that THs induced energy expenditure in response to increased caloric intake (3). Later, several intracellular processes were shown to be involved in the calorigenic effects of THs. These included increased ATP expenditure due to increased Na + /K + -ATPase activity to maintain ion gradients in various tissues (4, 5) as well as reduced efficiency of ATP synthesis, particularly through the induction of uncoupling proteins (UCPs), which cause proton leakage in the electron transport chain of the mitochondria of target tissues (6, 7). However, despite these advances in our understanding of THs on cellular metabolism, none of these proposed mechanisms appears to be dominant. Currently, little is known about other mechanisms that might be utilized by THs to regulate energy consumption within the cell. This is particularly true for the events involved in the delivery of fatty acids to mitochondria, a necessary step in converting stored intracellular triglyceride fuel into ATP.The active form of TH, 3,3′5-triiodo-l-thyronine (T 3 ), is a critical regulator of cellular and tissue metabolism throughout the body. It controls gene expression in target tissues by binding to its cognate nuclear receptors (TRα and TRβ), which are ligand-inducible transcription factors. In the presence of T 3 , TH receptors (TRs) bind to TH response elements in the promoters of target genes and form coactivator complexes containing histone acetyltransferase activity to activate transcription (8). In the absence of T 3 , TRs recruit corepressors such as NCoR and silencing mediator of retinoid and thyroid receptors (SMRT), which together with transducin β-like protein 1 (TBL1) and histone deacetylase 3 (HDAC3)

The dopamine transporter (DAT) modulates dopamine neurotransmission and is a primary target for psychostimulant influences on locomotion and reward. Selective DAT expression by dopaminergic neurons has led to use of cocaine analog DAT radioligands to assess rates of progression of dopamine neuronal degeneration in Parkinson's disease. We have documented that DAT is a phosphoprotein that is regulated by phosphorylation through pathways that include protein kinase C cascades. We now extend this work using drugs selective for phosphatidylinositol 3-kinase (PI3K), protein kinase C, MEK1/2, p38 kinase, and Ca 2؉ /calmodulin kinase II. We compare the drug effects on wild type DAT to the effects on 20 DAT mutants and a DAT deletion. PI3K and MEK1/2 modulators exert strong effects on DAT expression patterns and dopamine uptake V max . PKC principally modulates V max . Neither p38 nor Ca 2؉ /calmodulin kinase II agents exert significant influences on wild type DAT. Several mutants and a DAT with an N-terminal deletion display alterations that interact with the effects of kinase modulators, especially S7A for PKC effects; T62A, S581A, and T612A for PI3K effects; and S12A and T595A mutants for MEK1/2 effects. 32 P-Labeling studies confirm several of these effects of kinase pathway modulators on DAT phosphorylation. DAT expression and activities can be regulated by kinase cascades that require phosphoacceptor sites most concentrated in its N terminus. These results have a number of implications for DAT regulation and mandate caution in using DAT radioligand binding to infer changes in dopaminergic neuronal integrity after treatments that alter activities of these kinase pathways.The plasma membrane dopamine transporter (DAT) 1 functions to terminate dopaminergic neurotransmission by re-uptake of synaptic dopamine molecules into presynaptic neurons. Modulators of DAT function thus regulate the intensity and duration of dopaminergic neurotransmission (1, 2). DAT blockade by psychostimulants plays a key role in their rewarding and locomotor-stimulating properties (1, 3, 4). DAT uptake of dopamine-selective neurotoxins such as 6-hydroxydopamine and 1-methyl-4-phenylpyridinium is central to the cell type specificity of these model toxins for parkinsonian neurodegeneration.DAT is the most selective current marker for selected populations of dopaminergic neurons. It is expressed only in dopaminergic cells, with relatively high levels of expression in substantia nigra pars compacta neurons, intermediate levels in ventral tegmental area dopaminergic neurons, and low levels in arcuate nucleus neurons. These features, and the high affinity with which DAT can recognize cocaine analogs, have led to use of in vitro and in vivo assessments of DAT binding to seek evidence for degeneration of dopaminergic systems (5). DAT functions including its velocity of dopamine transport and its expression on plasma membranes can be regulated by several kinase or phosphatase pathways. DAT activity can be altered by activation of protein kinase C (PKC),...

Purpose of review Thyroid hormone (3,3′,5-triiodo-l-thyronine) plays an important role in thermogenesis and maintenance of lipid homeostasis. The present article reviews the evidence that 3,3′,5-triiodo-l-thyronine regulates lipid metabolism via thyroid hormone receptors, focusing particularly on in-vivo findings using genetically engineered mice. Recent findings That lipid metabolism is regulated via thyroid hormone receptor isoforms in a tissue-dependent manner was recently uncovered by using knockin mutant mice harboring an identical mutation in the Thra gene (Thra1PV mouse) or the Thrb gene (ThrbPV mouse). The mutation in the Thra gene dramatically decreases the mass of both white adipose tissue and liver. In contrast, the mutation in the Thrb gene markedly increases the mass of liver with an excess depot of lipids, but no significant abnormality is observed in white adipose tissue. Molecular studies show that the expression of lipogenic genes is decreased in white adipose tissue of Thra1PV mice, but not in ThrbPV mice. Markedly increased lipogenic enzyme expression, and decreased fatty acid beta-oxidation activity contribute to the adipogenic steatosis and lipid accumulation in the liver of ThrbPV mice. In contrast, reduced expression of genes critical for lipogenesis mediates decreased liver mass with lipid scarcity in Thra1PV mice. Summary Studies using Thra1PV and ThrbPV mice indicate that apo-thyroid hormone receptor-beta and apo-thyroid hormone receptor-alpha-1 mediate distinct deleterious effects on lipid metabolism. Thus, both thyroid hormone receptor isoforms contribute to the pathogenesis of lipid abnormalities in hypothyroidism, but in a target tissue-dependent manner. These studies suggest that thyroid hormone receptor isoform-specific ligands could be designed as therapeutic targets for lipid abnormalities.

Thyroid hormone receptors (TRs) play critical roles in energy homeostasis. To understand the role of TRs in lipid homeostasis in vivo, we adopted the loss-of-function approach by creating knock-in mutant mice with targeted mutation in the TRalpha gene (TRalpha1PV mouse) or TRbeta gene (TRbetaPV mouse). The PV mutation, identified in a patient with resistance to thyroid hormone, exhibits potent dominant-negative activity. Here we show that in contrast to TRalpha1PV mouse, TRbetaPV mice exhibited no significant reduction in WAT but had significant increases in serum free fatty acids and total triglycerides. Moreover, the liver of TRbetaPV mice was markedly increased (33%) with excess lipid accumulation, but the liver mass of TRalpha1PV mouse was decreased (23%) with paucity of lipids. These results indicate that apo-TRbeta and apo-TRalpha1 exerted distinct abnormalities in lipid metabolism. Further biochemical analyses indicate that increased lipogenic enzyme expression, activated peroxisome proliferator-activated receptor gamma (Ppargamma) signaling, and decreased fatty acid beta-oxidation activity contributed to the adipogenic steatosis and lipid accumulation in the liver of TRbetaPV mice. In contrast, the expression of lipogenic enzymes and Ppargamma was decreased in the liver of TRalpha1PV mice. These results suggest that the regulation of genes critical for lipid metabolism by TRs in the liver is isoform dependent. These results indicate that apo-TRbeta and apo-TRalpha1 had different effects on lipid metabolism and that both TR isoforms contribute to the pathogenesis of lipid metabolism in hypothyroidism.

Thyroid hormone nuclear receptors (TRs) are liganddependent transcription factors that regulate growth, differentiation, and development. To understand the role of the hormone, 3,3,5-triiodo-L-thyronine (T 3 ), in the nuclear translocation and targeting of TRs to the regulatory sites in chromatin, we appended green fluorescent protein (GFP) to the human TR subtype ␤1 (TR␤1). The fusion of GFP to the amino terminus of TR␤1 protein did not alter T 3 binding or transcriptional activities of the receptor. The subcellular localization of GFP-TR␤1 in living cells was visualized by laser-scanning confocal microscopy. In the presence of T 3 , the expressed GFP-TR␤1 was predominately localized in the nucleus, exhibiting a nuclear/cytoplasmic ratio of ϳ5.5. No GFP-TR␤1 was detected in the nucleolus. In the absence of T 3 , more GFP-TR␤1 was present in the cytoplasm, exhibiting a nuclear/cytoplasmic ratio of ϳ1.5. In these cells, cytoplasmic GFP-TR␤1 could be induced to enter the nucleus by T 3 . The T 3 -induced translocation was blocked when Lys 184 -Arg 185 in domain D of TR␤1 was mutated to Ala 184 -Ala 185 . Furthermore, the inability of the mutant TR to translocate to the nucleus correlated with the loss of most of its transcriptional activity. These results suggest that TR functions may, in part, be regulated by T 3 -induced nuclear entry.Thyroid hormone receptors (TRs) 1 are ligand-dependent transcription factors, which are members of the steroid hormone/retinoic acid receptor superfamily. Two TR genes, TR␣ and TR␤, located on chromosomes 17 and 3, respectively, give rise to four TR isoforms, ␣1, ␣2, ␤1, and ␤2, by alternative splicing of the primary transcripts (1, 2). TRs mediate the biological activities of the thyroid hormone, 3,3Ј,5-triiodo-Lthyronine (T 3 ), by binding to the specific DNA sequences, known as the thyroid hormone response elements (TREs), in the promoter regions of T 3 target genes (1, 2). The transcriptional regulatory activity of TRs not only depends on T 3 and the types of TREs but also on a host of co-regulatory proteins including co-repressors, co-activators, and the tumor suppressor p53 (3, 4).To function as transcription factors, TRs have to interact with transcriptional machinery in the nucleus. However, the process by which TRs are targeted to the nucleus is poorly understood. Before the genes encoding TRs were isolated, high affinity, low capacity T 3 binding sites were detected in the nuclear fractions of tissues and cultured cells by subcellular fractionation (5-9). Subsequently, when anti-TR antibodies became available, TRs were found only in the nuclei of fixed cells by immunocytochemistry and immunohistochemistry (10 -12). However, in these studies, dynamic cytoplasmic nuclear trafficking of TRs and the effect of T 3 on nuclear trafficking were not addressed. In the present study, we appended the green fluorescence protein (GFP) to the human TR subtype ␤1 (TR␤1) allowing direct examination of the nuclear transport of TRs in living cells. TR exhibited both constitutive nuclear l...

That a knock-in mouse harboring a dominant-negative thyroid hormone receptor (TR)-β (Thrb) mutation develops metastatic thyroid cancer strongly suggests the involvement of TRβ in carcinogenesis. Epigenetic silencing of the THRB gene is common in human cancers. The aim of the present study was to determine how DNA methylation affected the expression of the THRB gene in differentiated thyroid cancer (DTC) and how reexpression of the THRB gene attenuated the cancer phenotypes. We used methylation-specific PCR to examine the expression and promoter methylation of the THRB gene in DTC tissues. Thyroid cancer cells with hypermethylated THRB were treated with the demethylating agents 5'-aza-2'-deoxycytidine (5'-aza-CdR) and zebularine to evaluate their impact on the cancer cell phenotypes. THRB mRNA expression in DTC was 90% lower than in normal controls, and this decrease was associated with a higher tumor/lymph node staging. The promoter methylation level of the THRB gene had a significant negative correlation with the expression level of the THRB gene. Treatment of FTC-236 cells with 5'-aza-CdR or zebularine induced reexpression of the THRB gene and inhibited cell proliferation and migration. FTC-236 cells stably expressing TRβ exhibited lower cell proliferation and migration through inhibition of β-catenin signaling pathways compared with FTC-236 without TRβ. 5'-Aza-CdR also led to suppression of tumor growth in an in vivo xenograft model using FTC-236 cells consistent with the cell-based studies. These finding indicate that TRβ is a tumor suppressor and could be tested as a potential therapeutic target.

Background: Thyroid cancer is the most common endocrine tumor and is increasing in incidence. The aim of this study was to review mouse models of differentiated thyroid cancer and how they elucidate human thyroid cancer biology. Summary: Differentiated thyroid cancer, primarily papillary and follicular, comprises the majority of thyroid cancers. There has been tremendous growth in the cross-talk between basic science and clinical practice for thyroid cancer management. Insight into the framework of genes responsible for differentiated thyroid cancer has been gained through the use of mouse models. Common genetic alterations found in human papillary thyroid cancer such as RET=PTC rearrangements or the BRAF V600E mutation have genetically modified mouse counterparts. These and other preclinical mouse models have validated the importance of the cyclic adenosine monophosphate (cAMP)=protein kinase A and mitogen-activated protein kinase (MAPK) signaling pathways in papillary thyroid cancer (PTC). RAS mutations have a role in both papillary and follicular thyroid cancer development. Mice with overactivation of the phosphatidylinol-3-kinase (PI3K)-AKT and=or thyrotropinregulated signaling pathways have been found to develop follicular thyroid cancer. Additional mouse models of thyroid cancer that utilize inducible expression systems are in development or are being characterized and will better reflect the majority of human thyroid cancers which are non-hereditary. Advances in in vivo imaging of mice allow for earlier detection of metastasis and the ability to follow tumor growth or regression which may be used in evaluation of pharmaceutical agents. Conclusions: Mouse models have expanded our understanding of the altered signaling pathways that contribute to thyroid cancer tumorigenesis and provide a powerful tool to develop novel diagnostic approaches and therapies.

Purpose New therapeutic approaches are needed for patients with thyroid cancer refractory to radioiodine treatment. An inhibitor of bromodomain and extraterminal domain (BET) proteins, JQ1, shows potent anti-tumor effects in hematological cancers and solid tumors. To evaluate whether JQ1 is effective against thyroid cancer, we examined anti-tumor efficacy of JQ1 using the ThrbPV/PVKrasG12D mouse, a model of anaplastic thyroid cancer. Experimental Design We treated ThrbPV/PVKrasG12D mice with vehicle or JQ1 at a dose of 50 mg/kg body weight/day starting at the age of 8 weeks for a 10-week period and monitored thyroid tumor progression. Results JQ1 markedly inhibited thyroid tumor growth and prolonged survival of these mice. Global differential gene expression analysis showed that JQ1 suppressed the cMyc (hereafter referred to as Myc) transcription program by inhibiting mRNA expression of Myc, ccnd1, and other related genes. JQ1-suppressed Myc expression was accompanied by chromatin remodeling as evidenced by increased expression of histones and hexamethylene bis-acetamide inducible 1, a suppressor of RNA polymerase II transcription elongation. Analyses showed that JQ1 decreased MYC abundance in thyroid tumors and attenuated the cyclin-CDK4-Rb-E2F3 signaling to decrease tumor growth. Further analysis indicated that JQ1 inhibited the recruitment of BDR4 to the promoter complex of the Myc and Ccnd1 genes in rat thyroid follicular PCCL3 cells, resulting in decreased MYC expression at the mRNA and protein levels to inhibit tumor cell proliferation. Conclusions These preclinical findings suggest that BET inhibitors may be an effective agent to reduce thyroid tumor burden for the treatment of refractory thyroid cancer.