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1.
Nat Commun ; 10(1): 3700, 2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31420552

RESUMO

Little is known about the role of islet delta cells in regulating blood glucose homeostasis in vivo. Delta cells are important paracrine regulators of beta cell and alpha cell secretory activity, however the structural basis underlying this regulation has yet to be determined. Most delta cells are elongated and have a well-defined cell soma and a filopodia-like structure. Using in vivo optogenetics and high-speed Ca2+ imaging, we show that these filopodia are dynamic structures that contain a secretory machinery, enabling the delta cell to reach a large number of beta cells within the islet. This provides for efficient regulation of beta cell activity and is modulated by endogenous IGF-1/VEGF-A signaling. In pre-diabetes, delta cells undergo morphological changes that may be a compensation to maintain paracrine regulation of the beta cell. Our data provides an integrated picture of how delta cells can modulate beta cell activity under physiological conditions.


Assuntos
Ilhotas Pancreáticas/ultraestrutura , Comunicação Parácrina , Estado Pré-Diabético/patologia , Pseudópodes/ultraestrutura , Células Secretoras de Somatostatina/ultraestrutura , Animais , Glicemia/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/ultraestrutura , Microscopia Intravital , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Imagem Óptica , Optogenética , Estado Pré-Diabético/metabolismo , Pseudópodes/metabolismo , Células Secretoras de Somatostatina/citologia , Células Secretoras de Somatostatina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
2.
Cell Metab ; 30(2): 343-351.e3, 2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31178361

RESUMO

Most neurons are not replaced during an animal's lifetime. This nondividing state is characterized by extreme longevity and age-dependent decline of key regulatory proteins. To study the lifespans of cells and proteins in adult tissues, we combined isotope labeling of mice with a hybrid imaging method (MIMS-EM). Using 15N mapping, we show that liver and pancreas are composed of cells with vastly different ages, many as old as the animal. Strikingly, we also found that a subset of fibroblasts and endothelial cells, both known for their replicative potential, are characterized by the absence of cell division during adulthood. In addition, we show that the primary cilia of beta cells and neurons contains different structural regions with vastly different lifespans. Based on these results, we propose that age mosaicism across multiple scales is a fundamental principle of adult tissue, cell, and protein complex organization.

3.
J Cell Biol ; 218(2): 433-444, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30552100

RESUMO

Many adult tissues contain postmitotic cells as old as the host organism. The only organelle that does not turn over in these cells is the nucleus, and its maintenance represents a formidable challenge, as it harbors regulatory proteins that persist throughout adulthood. Here we developed strategies to visualize two classes of such long-lived proteins, histones and nucleoporins, to understand the function of protein longevity in nuclear maintenance. Genome-wide mapping of histones revealed specific enrichment of long-lived variants at silent gene loci. Interestingly, nuclear pores are maintained by piecemeal replacement of subunits, resulting in mosaic complexes composed of polypeptides with vastly different ages. In contrast, nondividing quiescent cells remove old nuclear pores in an ESCRT-dependent manner. Our findings reveal distinct molecular strategies of nuclear maintenance, linking lifelong protein persistence to gene regulation and nuclear integrity.


Assuntos
Regulação da Expressão Gênica/fisiologia , Histonas/metabolismo , Mitose/fisiologia , Poro Nuclear/metabolismo , Animais , Linhagem Celular , Estudo de Associação Genômica Ampla , Camundongos , Fatores de Tempo
4.
Nat Med ; 24(1): 39-49, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29200204

RESUMO

Thyroid hormone (TH) is critical for the maintenance of cellular homeostasis during stress responses, but its role in lung fibrosis is unknown. Here we found that the activity and expression of iodothyronine deiodinase 2 (DIO2), an enzyme that activates TH, were higher in lungs from patients with idiopathic pulmonary fibrosis than in control individuals and were correlated with disease severity. We also found that Dio2-knockout mice exhibited enhanced bleomycin-induced lung fibrosis. Aerosolized TH delivery increased survival and resolved fibrosis in two models of pulmonary fibrosis in mice (intratracheal bleomycin and inducible TGF-ß1). Sobetirome, a TH mimetic, also blunted bleomycin-induced lung fibrosis. After bleomycin-induced injury, TH promoted mitochondrial biogenesis, improved mitochondrial bioenergetics and attenuated mitochondria-regulated apoptosis in alveolar epithelial cells both in vivo and in vitro. TH did not blunt fibrosis in Ppargc1a- or Pink1-knockout mice, suggesting dependence on these pathways. We conclude that the antifibrotic properties of TH are associated with protection of alveolar epithelial cells and restoration of mitochondrial function and that TH may thus represent a potential therapy for pulmonary fibrosis.


Assuntos
Mitocôndrias/fisiologia , Fibrose Pulmonar/prevenção & controle , Hormônios Tireóideos/fisiologia , Animais , Células Cultivadas , Epitélio/fisiologia , Feminino , Humanos , Iodeto Peroxidase/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mimetismo Molecular , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Proteínas Quinases/genética , Fibrose Pulmonar/fisiopatologia
5.
Cell Rep ; 20(6): 1490-1501, 2017 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-28793270

RESUMO

Blood flow regulation in pancreatic islets is critical for function but poorly understood. Here, we establish an in vivo imaging platform in a non-human primate where islets transplanted autologously into the anterior chamber of the eye are monitored non-invasively and longitudinally at single-cell resolution. Engrafted islets were vascularized and innervated and maintained the cytoarchitecture of in situ islets in the pancreas. Blood flow velocity in the engrafted islets was not affected by increasing blood glucose levels and/or the GLP-1R agonist liraglutide. However, islet blood flow was dynamic in nature and fluctuated in various capillaries. This was associated with vasoconstriction events resembling a sphincter-like action, most likely regulated by adrenergic signaling. These observations suggest a mechanism in primate islets that diverts blood flow to cell regions with higher metabolic demand. The described imaging technology applied in non-human primate islets may contribute to a better understanding of human islet pathophysiology.


Assuntos
Velocidade do Fluxo Sanguíneo , Ilhotas Pancreáticas/irrigação sanguínea , Animais , Glicemia/metabolismo , Capilares/fisiologia , Células Cultivadas , Hipoglicemiantes/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Liraglutida/farmacologia , Macaca fascicularis , Masculino , Fluxo Sanguíneo Regional , Vasoconstrição
6.
Sci Rep ; 7: 44261, 2017 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-28303903

RESUMO

Thanks to the development of new 3D Imaging techniques, volumetric data of thick samples, especially tissues, are commonly available. Several algorithms were proposed to analyze cells or nuclei in tissues, however these tools are limited to two dimensions. Within any given tissue, cells are not likely to be organized randomly and as such have specific patterns of cell-cell interaction forming complex communication networks. In this paper, we propose a new set of tools as an approach to segment and analyze tissues in 3D with single cell resolution. This new tool box can identify and compute the geographical location of single cells and analyze the potential physical interactions between different cell types and in 3D. As a proof-of-principle, we applied our methodology to investigation of the cyto-architecture of the islets of Langerhans in mice and monkeys. The results obtained here are a significant improvement in current methodologies and provides new insight into the organization of alpha cells and their cellular interactions within the islet's cellular framework.


Assuntos
Algoritmos , Núcleo Celular/ultraestrutura , Processamento de Imagem Assistida por Computador/métodos , Ilhotas Pancreáticas/citologia , Análise de Célula Única/métodos , Animais , Comunicação Celular , Núcleo Celular/metabolismo , Expressão Gênica , Glucagon/genética , Glucagon/metabolismo , Haplorrinos , Processamento de Imagem Assistida por Computador/estatística & dados numéricos , Imagem Tridimensional , Insulina/genética , Insulina/metabolismo , Ilhotas Pancreáticas/anatomia & histologia , Camundongos , Camundongos Endogâmicos C57BL , Imagem Óptica/métodos , Somatostatina/genética , Somatostatina/metabolismo
7.
Diabetologia ; 58(10): 2218-28, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26215305

RESUMO

The human genome project and its search for factors underlying human diseases has fostered a major human research effort. Therefore, unsurprisingly, in recent years we have observed an increasing number of studies on human islet cells, including disease approaches focusing on type 1 and type 2 diabetes. Yet, the field of islet and diabetes research relies on the legacy of rodent-based investigations, which have proven difficult to translate to humans, particularly in type 1 diabetes. Whole islet physiology and pathology may differ between rodents and humans, and thus a comprehensive cross-species as well as species-specific view on islet research is much needed. In this review we summarise the current knowledge of interspecies islet cytoarchitecture, and discuss its potential impact on islet function and future perspectives in islet pathophysiology research.


Assuntos
Ilhotas Pancreáticas/anatomia & histologia , Ilhotas Pancreáticas/fisiologia , Animais , Humanos , Especificidade da Espécie
8.
Proc Natl Acad Sci U S A ; 111(49): 17612-7, 2014 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-25404292

RESUMO

Pancreatic islets secrete hormones that play a key role in regulating blood glucose levels (glycemia). Age-dependent impairment of islet function and concomitant dysregulation of glycemia are major health threats in aged populations. However, the major causes of the age-dependent decline of islet function are still disputed. Here we demonstrate that aging of pancreatic islets in mice and humans is notably associated with inflammation and fibrosis of islet blood vessels but does not affect glucose sensing and the insulin secretory capacity of islet beta cells. Accordingly, when transplanted into the anterior chamber of the eye of young mice with diabetes, islets from old mice are revascularized with healthy blood vessels, show strong islet cell proliferation, and fully restore control of glycemia. Our results indicate that beta cell function does not decline with age and suggest that islet function is threatened by an age-dependent impairment of islet vascular function. Strategies to mitigate age-dependent dysregulation in glycemia should therefore target systemic and/or local inflammation and fibrosis of the aged islet vasculature.


Assuntos
Envelhecimento , Glicemia/metabolismo , Capilares/fisiologia , Ilhotas Pancreáticas/fisiologia , Adolescente , Adulto , Idoso , Animais , Proliferação de Células , Fibrose , Glucose/metabolismo , Homeostase , Humanos , Inflamação , Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Perfusão , Fatores de Tempo , Adulto Jovem
9.
Mol Endocrinol ; 27(12): 2105-15, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24196352

RESUMO

The type II iodothyronine deiodinase (D2) is a type I endoplasmic reticulum (ER)-resident thioredoxin fold-containing selenoprotein that activates thyroid hormone. D2 is inactivated by ER-associated ubiquitination and can be reactivated by two ubiquitin-specific peptidase-class D2-interacting deubiquitinases (DUBs). Here, we used D2-expressing cell models to define that D2 ubiquitination (UbD2) occurs via K48-linked ubiquitin chains and that exposure to its natural substrate, T4, accelerates UbD2 formation and retrotranslocation to the cytoplasm via interaction with the p97-ATPase complex. D2 retrotranslocation also includes deubiquitination by the p97-associated DUB Ataxin-3 (Atx3). Inhibiting Atx3 with eeyarestatin-I did not affect D2:p97 binding but decreased UbD2 retrotranslocation and caused ER accumulation of high-molecular weight UbD2 bands possibly by interfering with the D2-ubiquitin-specific peptidases binding. Once in the cytosol, D2 is delivered to the proteasomes as evidenced by coprecipitation with 19S proteasome subunit S5a and increased colocalization with the 20S proteasome. We conclude that interaction between UbD2 and p97/Atx3 mediates retranslocation of UbD2 to the cytoplasm for terminal degradation in the proteasomes, a pathway that is accelerated by exposure to T4.


Assuntos
Adenosina Trifosfatases/metabolismo , Citoplasma/enzimologia , Iodeto Peroxidase/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Repressoras/metabolismo , Ataxina-3 , Retículo Endoplasmático/metabolismo , Degradação Associada com o Retículo Endoplasmático , Estabilidade Enzimática , Células HEK293 , Humanos , Lisina/metabolismo , Transporte Proteico , Especificidade por Substrato , Ubiquitina/metabolismo , Ubiquitinação
10.
J Clin Invest ; 123(4): 1492-500, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23524969

RESUMO

Type II deiodinase (D2) activates thyroid hormone by converting thyroxine (T4) to 3,5,3'-triiodothyronine (T3). This allows plasma T4 to signal a negative feedback loop that inhibits production of thyrotropin-releasing hormone (TRH) in the mediobasal hypothalamus (MBH) and thyroid-stimulating hormone (TSH) in the pituitary. To determine the relative contributions of these D2 pathways in the feedback loop, we developed 2 mouse strains with pituitary- and astrocyte-specific D2 knockdown (pit-D2 KO and astro-D2 KO mice, respectively). The pit-D2 KO mice had normal serum T3 and were systemically euthyroid, but exhibited an approximately 3-fold elevation in serum TSH levels and a 40% reduction in biological activity. This was the result of elevated serum T4 that increased D2-mediated T3 production in the MBH, thus decreasing Trh mRNA. That tanycytes, not astrocytes, are the cells within the MBH that mediate T4-to-T3 conversion was defined by studies using the astro-D2 KO mice. Despite near-complete loss of brain D2, tanycyte D2 was preserved in astro-D2 KO mice at levels that were sufficient to maintain both the T4-dependent negative feedback loop and thyroid economy. Taken together, these data demonstrated that the hypothalamic-thyroid axis is wired to maintain normal plasma T3 levels, which is achieved through coordination of T4-to-T3 conversion between thyrotrophs and tanycytes.


Assuntos
Regulação da Expressão Gênica , Hipotálamo/enzimologia , Iodeto Peroxidase/metabolismo , Hipófise/enzimologia , Tireotropina/genética , Tri-Iodotironina/sangue , Animais , Astrócitos/enzimologia , Composição Corporal , Córtex Cerebral/metabolismo , Ativação Enzimática , Retroalimentação Fisiológica , Proteína Glial Fibrilar Ácida/metabolismo , Hipocampo/metabolismo , Hipotálamo/citologia , Hipotálamo/metabolismo , Iodeto Peroxidase/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Especificidade de Órgãos , Hipófise/citologia , Glândula Tireoide/metabolismo , Glândula Tireoide/fisiologia , Tireotrofos/enzimologia , Tireotropina/sangue , Hormônio Liberador de Tireotropina , Tiroxina/sangue , Tiroxina/fisiologia , Tri-Iodotironina/fisiologia
11.
Biochim Biophys Acta ; 1830(7): 3956-64, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22967761

RESUMO

BACKGROUND: Thyroid hormone signaling is critical for development, growth and metabolic control in vertebrates. Although serum concentration of thyroid hormone is remarkable stable, deiodinases modulate thyroid hormone signaling on a time- and cell-specific fashion by controlling the activation and inactivation of thyroid hormone. SCOPE OF THE REVIEW: This review covers the recent advances in D2 biology, a member of the iodothyronine deiodinase family, thioredoxin fold-containing selenoenzymes that modify thyroid hormone signaling in a time- and cell-specific manner. MAJOR CONCLUSIONS: D2-catalyzed T3 production increases thyroid hormone signaling whereas blocking D2 activity or disruption of the Dio2 gene leads to a state of localized hypothyroidism. D2 expression is regulated by different developmental, metabolic or environmental cues such as the hedgehog pathway, the adrenergic- and the TGR5-activated cAMP pathway, by xenobiotic molecules such as flavonols and by stress in the endoplasmic reticulum, which specifically reduces de novo synthesis of D2 via an eIF2a-mediated mechanism. Thus, D2 plays a central role in important physiological processes such as determining T3 content in developing tissues and in the adult brain, and promoting adaptive thermogenesis in brown adipose tissue. Notably, D2 is critical in the T4-mediated negative feed-back at the pituitary and hypothalamic levels, whereby T4 inhibits TSH and TRH expression, respectively. Notably, ubiquitination is a major step in the control of D2 activity, whereby T4 binding to and/or T4 catalysis triggers D2 inactivation by ubiquitination that is mediated by the E3 ubiquitin ligases WSB-1 and/or TEB4. Ubiquitinated D2 can be either targeted to proteasomal degradation or reactivated by deubiquitination, a process that is mediated by the deubiquitinases USP20/33 and is important in adaptive thermogenesis. GENERAL SIGNIFICANCE: Here we review the recent advances in the understanding of D2 biology focusing on the mechanisms that regulate its expression and their biological significance in metabolically relevant tissues. This article is part of a Special Issue entitled Thyroid hormone signalling.


Assuntos
Iodeto Peroxidase/metabolismo , Hormônios Tireóideos/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Iodeto Peroxidase/genética , Transdução de Sinais , Hormônios Tireóideos/genética
12.
J Neurosci ; 32(25): 8491-500, 2012 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-22723689

RESUMO

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.


Assuntos
Hipóxia Celular/fisiologia , Núcleo Celular/metabolismo , Proteínas de Choque Térmico HSP40/fisiologia , Iodeto Peroxidase/metabolismo , Neurônios/metabolismo , Animais , Isquemia Encefálica/metabolismo , Núcleo Celular/enzimologia , Células Cultivadas , DNA/genética , Retículo Endoplasmático/metabolismo , Glicosilação , Hipocampo/citologia , Hipocampo/metabolismo , Imuno-Histoquímica , Imunoprecipitação , Masculino , Microscopia Eletrônica , Artéria Cerebral Média/fisiologia , Consumo de Oxigênio/fisiologia , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-Dawley , Receptores dos Hormônios Tireóideos/metabolismo , Transdução de Sinais/fisiologia , Hormônios Tireóideos/fisiologia
13.
PLoS One ; 7(6): e37860, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22719854

RESUMO

Hypothalamic neurosecretory systems are fundamental regulatory circuits influenced by thyroid hormone. Monocarboxylate-transporter-8 (MCT8)-mediated uptake of thyroid hormone followed by type 3 deiodinase (D3)-catalyzed inactivation represent limiting regulatory factors of neuronal T3 availability. In the present study we addressed the localization and subcellular distribution of D3 and MCT8 in neurosecretory neurons and addressed D3 function in their axons. Intense D3-immunoreactivity was observed in axon varicosities in the external zone of the rat median eminence and the neurohaemal zone of the human infundibulum containing axon terminals of hypophysiotropic parvocellular neurons. Immuno-electronmicroscopy localized D3 to dense-core vesicles in hypophysiotropic axon varicosities. N-STORM-superresolution-microscopy detected the active center containing C-terminus of D3 at the outer surface of these organelles. Double-labeling immunofluorescent confocal microscopy revealed that D3 is present in the majority of GnRH, CRH and GHRH axons but only in a minority of TRH axons, while absent from somatostatin-containing neurons. Bimolecular-Fluorescence-Complementation identified D3 homodimers, a prerequisite for D3 activity, in processes of GT1-7 cells. Furthermore, T3-inducible D3 catalytic activity was detected in the rat median eminence. Triple-labeling immunofluorescence and immuno-electronmicroscopy revealed the presence of MCT8 on the surface of the vast majority of all types of hypophysiotropic terminals. The presence of MCT8 was also demonstrated on the axon terminals in the neurohaemal zone of the human infundibulum. The unexpected role of hypophysiotropic axons in fine-tuned regulation of T3 availability in these cells via MCT8-mediated transport and D3-catalyzed inactivation may represent a novel regulatory core mechanism for metabolism, growth, stress and reproduction in rodents and humans.


Assuntos
Hipotálamo/fisiologia , Neurônios/fisiologia , Hormônios Tireóideos/metabolismo , Animais , Axônios , Imunofluorescência , Humanos , Hipotálamo/citologia , Hipotálamo/metabolismo , Imuno-Histoquímica , Masculino , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa
14.
Mol Endocrinol ; 25(12): 2065-75, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22053000

RESUMO

Cells respond rapidly to endoplasmic reticulum (ER) stress by blocking protein translation, increasing protein folding capacity, and accelerating degradation of unfolded proteins via ubiquitination and ER-associated degradation pathways. The ER resident type 2 deiodinase (D2) is normally ubiquitinated and degraded in the proteasome, a pathway that is accelerated by enzyme catalysis of T(4) to T(3). To test whether D2 is normally processed through ER-associated degradation, ER stress was induced in cells that endogenously express D2 by exposure to thapsigargin or tunicamycin. In all cell models, D2 activity was rapidly lost, to as low as of 30% of control activity, without affecting D2 mRNA levels; loss of about 40% of D2 activity and protein was also seen in human embryonic kidney 293 cells transiently expressing D2. In primary human airway cells with ER stress resulting from cystic fibrosis, D2 activity was absent. The rapid ER stress-induced loss of D2 resulted in decreased intracellular D2-mediated T(3) production. ER stress-induced loss of D2 was prevented in the absence of T(4), by blocking the proteasome with MG-132 or by treatment with chemical chaperones. Notably, ER stress did not alter D2 activity half-life but rather decreased D2 synthesis as assessed by induction of D2 mRNA and by [(35)S]methionine labeling. Remarkably, ER-stress-induced loss in D2 activity is prevented in cells transiently expressing an inactive eukaryotic initiation factor 2, indicating that this pathway mediates the loss of D2 activity. In conclusion, D2 is selectively lost during ER stress due to an eukaryotic initiation factor 2-mediated decrease in D2 synthesis and sustained proteasomal degradation. This explains the lack of D2 activity in primary human airway cells with ER stress resulting from cystic fibrosis.


Assuntos
Estresse do Retículo Endoplasmático , Fator de Iniciação 2 em Eucariotos/metabolismo , Iodeto Peroxidase/metabolismo , Tiroxina/metabolismo , Tri-Iodotironina/biossíntese , Animais , Linhagem Celular , Fibrose Cística/enzimologia , Regulação para Baixo , Células Epiteliais/metabolismo , Expressão Gênica , Humanos , Iodeto Peroxidase/genética , Camundongos , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , Proteólise , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Transdução de Sinais , Tapsigargina , Fator de Transcrição CHOP/metabolismo , Tunicamicina
15.
Int J Biochem Cell Biol ; 43(10): 1432-41, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21679772

RESUMO

Thyroid hormone action can be customized on a cell-specific fashion through the controlled action of the deiodinase group of enzymes, which are homodimeric thioredoxin fold containing selenoproteins. Whereas the type II deiodinase (D2) initiates thyroid hormone signaling by activating the pro-hormone thyroxine (T4) to the biologically active T3 molecule, the type III deiodinase (D3) terminates thyroid hormone action by catalyzing the inactivation of both T4 and T3 molecules. Deiodinases play a role in thyroid hormone homeostasis, development, growth and metabolic control by affecting the intracellular levels of T3 and thus gene expression on a cell-specific basis. Whereas both Dio2 and Dio3 are transcriptionally regulated, ubiquitination of D2 is a switch mechanism that controls D2 activity and intracellular T3 production. The hedgehog-inducible WSB-1 and the yeast Doa10 mammalian ortholog TEB4 are two E3 ligases that inactivate D2 via ubiquitination. Inactivation involves disruption of the D2:D2 dimer and can be reversed via two ubiquitin-specific proteases, USP20 and USP33, rescuing catalytic activity and T3 production. The ubiquitin-based switch mechanism that controls D2 activity illustrates how different cell types fine-tune thyroid hormone signaling, making D2 a suitable target for pharmacological intervention. This article reviews the cellular and molecular aspects of D2 regulation and the current models of D2-mediated thyroid hormone signaling.


Assuntos
Iodeto Peroxidase/metabolismo , Tiroxina/metabolismo , Tri-Iodotironina/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Iodeto Peroxidase/genética , Camundongos , Complexo de Endopeptidases do Proteassoma/metabolismo , Transdução de Sinais , Tiroxina/genética , Distribuição Tecidual , Tri-Iodotironina/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
16.
FEBS Lett ; 585(3): 539-44, 2011 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-21237159

RESUMO

Exposure of cell lines endogenously expressing the thyroid hormone activating enzyme type 2 deiodinase (D2) to the chemical chaperones tauroursodeoxycholic acid (TUDCA) or 4-phenylbutiric acid (4-PBA) increases D2 expression, activity and T3 production. In brown adipocytes, TUDCA or 4-PBA induced T3-dependent genes and oxygen consumption (∼2-fold), an effect partially lost in D2 knockout cells. In wild type, but not in D2 knockout mice, administration of TUDCA lowered the respiratory quotient, doubled brown adipose tissue D2 activity and normalized the glucose intolerance associated with high fat feeding. Thus, D2 plays a critical role in the metabolic effects of chemical chaperones.


Assuntos
Metabolismo Energético/efeitos dos fármacos , Iodeto Peroxidase/metabolismo , Fenilbutiratos/farmacologia , Ácido Tauroquenodesoxicólico/farmacologia , Tri-Iodotironina/metabolismo , Adipócitos Marrons/efeitos dos fármacos , Adipócitos Marrons/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Gorduras na Dieta/efeitos adversos , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Intolerância à Glucose/prevenção & controle , Humanos , Iodeto Peroxidase/genética , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Consumo de Oxigênio/efeitos dos fármacos , RNA Mensageiro/metabolismo
17.
Endocrinology ; 151(12): 5961-70, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20926587

RESUMO

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


Assuntos
Amiodarona/efeitos adversos , Antiarrítmicos/efeitos adversos , Iodeto Peroxidase/metabolismo , Tireotropina/sangue , Animais , Linhagem Celular , Iodeto Peroxidase/antagonistas & inibidores , Iodeto Peroxidase/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
18.
Endocrinology ; 150(11): 5171-80, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19819956

RESUMO

The type 3 iodothyronine deiodinase (D3) is the primary deiodinase that inactivates thyroid hormone. Immunoprecipitation of D3, followed by fluorescent two-dimensional difference gel electrophoresis and mass spectrometry, identified peroxiredoxin 3 (Prx3) as a D3-associated protein. This interaction was confirmed using reverse coimmunoprecipitation, in which pull-down of Prx3 resulted in D3 isolation, and by fluorescence resonance energy transfer between cyan fluorescent protein-D3 and yellow fluorescent protein-Prx3. Prx3 overexpression did not change D3 activity in transfected HEK 293 cells; however, Prx3 knockdown resulted in a 50% decrease in D3-mediated whole-cell deiodination. Notably, D3 activity of cell lysates with dithiothreitol as an exogenous reducing factor and D3 protein levels were not decreased with Prx3 knockdown, indicating that the observed reduction in whole-cell deiodination was not simply due to a decrease in D3 enzyme levels. Prx3 knockdown did not change D3's affinity for T3 because saturation of D3-mediated whole-cell deiodination occurred between 20 and 200 nm T3 both with and without Prx3. Furthermore, the decrease in D3 activity in whole cells was not attributable to nonspecific oxidative stress because pretreatment with the antioxidant N-acetyl cysteine did not reverse the effects of Prx3 knockdown. Thioredoxin, the cofactor needed for Prx3 regeneration, supported D3 microsomal activity; however, Prx3 knockdown did not change D3 activity in this system. In conclusion, knockdown of Prx3 decreases D3 activity in whole cells, whereas absolute levels of D3 are unchanged, consistent with Prx3 playing a rate-limiting role in the regeneration of the D3 enzyme.


Assuntos
Iodeto Peroxidase/metabolismo , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Linhagem Celular , Técnicas de Silenciamento de Genes , Halogenação , Humanos , Iodeto Peroxidase/genética , Ligação Proteica , Tri-Iodotironina/metabolismo
19.
Mol Cell Biol ; 29(19): 5339-47, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19651899

RESUMO

The endoplasmic reticulum resident thyroid hormone-activating type 2 deiodinase (D2) is inactivated by ubiquitination via the hedgehog-inducible WSB-1. Ubiquitinated D2 can then be subsequently taken up by the proteasomal system or be reactivated by USP-33/20-mediated deubiquitination. Given that heterologously expressed D2 accumulates in Saccharomyces cerevisiae lacking the E3 ligase Doa10, we tested whether the human Doa10 ortholog, TEB4, plays a role in D2 ubiquitination and degradation. In a setting of transient coexpression in HEK-293 cells, TEB4 and D2 could be coimmunoprecipitated, and additional TEB4 expression decreased D2 activity by approximately 50% (P < 0.05). A highly efficient TEB4 knockdown (>90% reduction in mRNA and protein levels) decreased D2 ubiquitination and increased D2 activity and protein levels by about fourfold. The other activating deiodinase, D1, or a truncated D2 molecule (Delta18-D2) that lacks a critical instability domain was not affected by TEB4 knockdown. Furthermore, TEB4 knockdown prolonged D2 activity half-life at least fourfold, even under conditions known to promote D2 ubiquitination. Neither exposure to 1 microM of the proteasomal inhibitor MG132 for 24 h nor RNA interference WSB-1 knockdown resulted in additive effects on D2 expression when combined with TEB4 knockdown. Similar results were obtained with MSTO-211 cells, which endogenously express D2, after TEB4 knockdown using a lentivirus-based transduction strategy. While TEB4 expression predominates in the hematopoietic lineage, both WSB-1 and TEB4 are coexpressed with D2 in a number of tissues and cell types, except the thyroid and brown adipose tissue, where TEB4 expression is minimal. We conclude that TEB4 interacts with and mediates loss of D2 activity, indicating that D2 ubiquitination and degradation can be tissue specific, depending on WSB-1 and TEB4 expression levels.


Assuntos
Iodeto Peroxidase/metabolismo , Proteínas de Membrana/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Encéfalo/enzimologia , Proteínas de Transporte/genética , Linhagem Celular , Ativação Enzimática , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Proteínas de Membrana/genética , Especificidade de Órgãos , Complexo de Endopeptidases do Proteassoma/metabolismo , Interferência de RNA , Ratos , Ratos Wistar , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
20.
Endocrinology ; 148(3): 954-60, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17138654

RESUMO

For T(3) to mediate its biological effects, the prohormone T(4) must be activated by removal of an outer-ring iodine by the type 1 or 2 deiodinases (D1 and D2) with approximately 60% of the daily T(3) production in rodents being produced extrathyroidally through this pathway. To further define the role of these enzymes in thyroid hormone homeostasis, we backcrossed the targeted disruption of the Dio2 gene into C3H/HeJ (C3H) mice with genetically low D1 expression to create the C3H-D2KO mouse. Remarkably, these mice maintain euthyroid serum T(3) levels with normal growth and no decrease in expression of hepatic T(3)-responsive genes. However, serum T(4) is increased 1.2-fold relative to the already elevated C3H levels, and serum TSH is increased 1.4-fold. Despite these increases, thyroidal (125)I uptake indicates no difference in thyroidal activity between C3H-D2KO and C3H mice. Although C3H-D2KO hepatic and renal D1 activities were well below those observed in wild-type mice (approximately 0.1-fold for both), they were 8-fold and 2-fold higher, respectively, relative to C3H mice. Thyroidal D1 and cerebral cortical type 3 deiodinase activity were unchanged between C3H-D2KO and C3H mice. In conclusion, C3H-D2KO mice have notably elevated serum T(4) levels, and this, in conjunction with residual D1 activity, is likely an important role in the maintenance of euthyroid serum T(3) concentrations.


Assuntos
Iodeto Peroxidase/genética , Tiroxina/metabolismo , Tri-Iodotironina/sangue , Tri-Iodotironina/metabolismo , Animais , Cruzamentos Genéticos , Feminino , Iodeto Peroxidase/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout
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