RESUMEN
CONTEXT: Lower TSH screening cutoffs have doubled the ascertainment of congenital hypothyroidism (CH), particularly cases with a eutopically located gland-in-situ (GIS). Although mutations in known dyshormonogenesis genes or TSHR underlie some cases of CH with GIS, systematic screening of these eight genes has not previously been undertaken. OBJECTIVE: Our objective was to evaluate the contribution and molecular spectrum of mutations in eight known causative genes (TG, TPO, DUOX2, DUOXA2, SLC5A5, SLC26A4, IYD, and TSHR) in CH cases with GIS. Patients, Design, and Setting: We screened 49 CH cases with GIS from 34 ethnically diverse families, using next-generation sequencing. Pathogenicity of novel mutations was assessed in silico. PATIENTS, DESIGN, AND SETTING: We screened 49 CH cases with GIS from 34 ethnically diverse families, using next-generation sequencing. Pathogenicity of novel mutations was assessed in silico. RESULTS: Twenty-nine cases harbored likely disease-causing mutations. Monogenic defects (19 cases) most commonly involved TG (12), TPO (four), DUOX2 (two), and TSHR (one). Ten cases harbored triallelic (digenic) mutations: TG and TPO (one); SLC26A4 and TPO (three), and DUOX2 and TG (six cases). Novel variants overall included 15 TG, six TPO, and three DUOX2 mutations. Genetic basis was not ascertained in 20 patients, including 14 familial cases. CONCLUSIONS: The etiology of CH with GIS remains elusive, with only 59% attributable to mutations in TSHR or known dyshormonogenesis-associated genes in a cohort enriched for familial cases. Biallelic TG or TPO mutations most commonly underlie severe CH. Triallelic defects are frequent, mandating future segregation studies in larger kindreds to assess their contribution to variable phenotype. A high proportion (â¼41%) of unsolved or ambiguous cases suggests novel genetic etiologies that remain to be elucidated.
Asunto(s)
Autoantígenos/genética , Hipotiroidismo Congénito/genética , Yoduro Peroxidasa/genética , Proteínas de Unión a Hierro/genética , Receptores de Tirotropina/genética , Tiroglobulina/genética , Humanos , Mutación , Linaje , FenotipoRESUMEN
In 2012, we learned that functional thyroid tissue can be generated in vitro, and that thyroid hormones stimulate autophagy. Patients with defects in TRα have been identified, and di-iodothyropropionic acid has been shown to ameliorate MCT8 deficiency. Finally, we found that gene expression profiling can identify benign thyroid nodules.
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Hipotiroidismo/terapia , Glándula Tiroides/citología , Glándula Tiroides/metabolismo , Hormonas Tiroideas/metabolismo , Neoplasias de la Tiroides/terapia , Animales , Autofagia , Humanos , Hipotiroidismo/tratamiento farmacológico , Hipotiroidismo/genética , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Receptores de Hormona Tiroidea/genética , Receptores de Hormona Tiroidea/metabolismo , Simportadores , Neoplasias de la Tiroides/diagnóstico , Neoplasias de la Tiroides/cirugíaRESUMEN
OBJECTIVE: In this study, we aimed to investigate the genetic background of thyroid dyshormonogenesis (TDH). CONTEXT: Thyroid dyshormonogenesis comprises 10-15% of all cases of congenital hypothyroidism (CH), which is the most common neonatal endocrine disorder, and might result from disruptions at any stage of thyroid hormone biosynthesis. Currently seven genes (NIS, TPO, PDS, TG, IYD, DUOX2 and DUOXA2) have been implicated in the aetiology of the disease. DESIGN: As TDH is mostly inherited in an autosomal recessive manner, we planned to conduct the study in consanguineous/multi-case families. PATIENTS: One hundred and four patients with congenital TDH all coming from consanguineous and/or multi-case families. MEASUREMENTS: Initially, we performed potential linkage analysis of cases to all seven causative-TDH loci as well as direct sequencing of the TPO gene in cases we could not exclude linkage to this locus. In addition, in silico analyses of novel missense mutations were carried out. RESULTS: TPO had the highest potential for linkage and we identified 21 TPO mutations in 28 TDH cases showing potential linkage to this locus. Four of 10 distinct TPO mutations detected in this study were novel (A5T, Y55X, E596X, D633N). CONCLUSIONS: This study underlines the importance of molecular genetic studies in diagnosis, classification and prognosis of CH and proposes a comprehensive mutation screening by new sequencing technology in all newly diagnosed primary CH cases.
Asunto(s)
Hipotiroidismo Congénito/genética , Consanguinidad , Yoduro Peroxidasa/genética , Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Mutación Missense , Pakistán , Hormonas Tiroideas/biosíntesis , Hormonas Tiroideas/genética , TurquíaRESUMEN
CONTEXT: Patients with Addison's disease (AD) self-report impairment in specific dimensions on well-being questionnaires. An AD-specific quality-of-life questionnaire (AddiQoL) was developed to aid evaluation of patients. OBJECTIVE: We aimed to translate and determine construct validity, reliability, and concurrent validity of the AddiQoL questionnaire. METHODS: After translation, the final versions were tested in AD patients from Norway (n = 107), Sweden (n = 101), Italy (n = 165), Germany (n = 200), and Poland (n = 50). Construct validity was examined by exploratory factor analysis and Rasch analysis, aiming at unidimensionality and fit to the Rasch model. Reliability was determined by Cronbach's coefficient-α and Person separation index. Longitudinal reliability was tested by differential item functioning in stable patient subgroups. Concurrent validity was examined in Norwegian (n = 101) and Swedish (n = 107) patients. RESULTS: Exploratory factor analysis and Rasch analysis identified six items with poor psychometric properties. The 30 remaining items fitted the Rasch model and proved unidimensional, supported by appropriate item and person fit residuals and a nonsignificant χ(2) probability. Crohnbach's α-coefficient 0.93 and Person separation index 0.86 indicate high reliability. Longitudinal reliability was excellent. Correlation with Short Form-36 and Psychological General Well-Being Index scores was high. A shorter subscale comprising eight items also proved valid and reliable. Testing of AddiQoL-30 in this large patient cohort showed significantly worse scores with increasing age and in women compared with men but no difference between patients with isolated AD and those with concomitant diseases. CONCLUSION: The validation process resulted in a revised 30-item AddiQoL questionnaire and an eight-item AddiQoL short version with good psychometric properties and high reliability.
Asunto(s)
Enfermedad de Addison/epidemiología , Enfermedad de Addison/psicología , Calidad de Vida , Encuestas y Cuestionarios , Enfermedad de Addison/diagnóstico , Enfermedad de Addison/fisiopatología , Adolescente , Adulto , Anciano , Europa (Continente) , Femenino , Alemania/epidemiología , Humanos , Italia/epidemiología , Masculino , Persona de Mediana Edad , Noruega/epidemiología , Polonia/epidemiología , Reproducibilidad de los Resultados , Suecia/epidemiología , Adulto JovenRESUMEN
CONTEXT: Patients with Addison's disease reproducibly self-report impairment in specific dimensions of general well-being questionnaires, suggesting particular deficiencies in health-related quality-of-life (HRQoL). OBJECTIVE: We sought to develop an Addison's disease-specific questionnaire (AddiQoL) that could better quantify altered well-being and treatment effects. Design, Setting, Patients, Intervention, and Outcomes: We reviewed the literature to identify HRQoL issues in Addison's disease and interviewed patients and their partners in-depth to explore various symptom domains. A list of items was generated, and nine expert clinicians and five expert patients assessed the list for impact and clarity. A preliminary questionnaire was presented to 100 Addison's outpatients; the number of items was reduced after analysis of the distribution of the responses. The final questionnaire responses were assessed by Cronbach's alpha and Rasch analysis. RESULTS AND INTERPRETATION: Published studies of HRQoL in Addison's disease indicated reduced vitality and general health perception and limitations in physical and emotional functioning. In-depth interviews of 14 patients and seven partners emphasized the impact of the disease on the emotional domain. Seventy HRQoL items were generated; after the expert consultation process and pretesting in 100 patients, the number of items was reduced to 36. Eighty-six patients completed the final questionnaire; the responses showed high internal consistency with Cronbach's alpha 0.95 and Person Separation Index 0.94 (Rasch analysis). CONCLUSIONS: We envisage AddiQoL having utility in trials of hormone replacement and management of patients with Addison's disease, analogous to similar questionnaires in GH deficiency (AGHDA) and acromegaly (AcroQoL).
Asunto(s)
Enfermedad de Addison/psicología , Calidad de Vida , Encuestas y Cuestionarios , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
UNLABELLED: Context Patients with primary adrenal insufficiency (Addison's disease) receive more glucococorticoids than the normal endogenous production, raising concern about adverse effects on bone. OBJECTIVE: To determine i) the effects of glucocorticoid replacement therapy on bone, and ii) the impact of glucocorticoid pharmacogenetics. DESIGN, SETTING AND PARTICIPANTS: A cross-sectional study of two large Addison's cohorts from Norway (n=187) and from UK and New Zealand (n=105). MAIN OUTCOME MEASURES: Bone mineral density (BMD) was measured; the Z-scores represent comparison with a reference population. Blood samples from 187 Norwegian patients were analysed for bone markers and common polymorphisms in genes that have been associated with glucocorticoid sensitivity. RESULTS: Femoral neck BMD Z-scores were significantly reduced in the patients (Norway: mean -0.28 (95% confidence intervals (CI) -0.42, -0.16); UK and New Zealand: -0.21 (95% CI -0.36, -0.06)). Lumbar spine Z-scores were reduced (Norway: -0.17 (-0.36, +0.01); UK and New Zealand: -0.57 (-0.78, -0.37)), and significantly lower in males compared with females (P=0.02). The common P-glycoprotein (ABCB1) polymorphism C3435T was significantly associated with total BMD (CC and CT>TT P=0.015), with a similar trend at the hip and spine. CONCLUSIONS: BMD at the femoral neck and lumbar spine is reduced in Addison's disease, indicating undesirable effects of the replacement therapy. The findings lend support to the recommendations that 15-25 mg hydrocortisone daily is more appropriate than the higher conventional doses. A common polymorphism in the efflux transporter P-glycoprotein is associated with reduced bone mass and might confer susceptibility to glucocorticoid induced osteoporosis.
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Enfermedad de Addison/tratamiento farmacológico , Enfermedad de Addison/genética , Huesos/metabolismo , Glucocorticoides/uso terapéutico , Terapia de Reemplazo de Hormonas/métodos , Farmacogenética , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Adolescente , Adulto , Anciano , Densidad Ósea , Estudios de Cohortes , Estudios Transversales , Femenino , Humanos , Hidrocortisona/uso terapéutico , Masculino , Persona de Mediana Edad , Nueva Zelanda , Polimorfismo Genético/genética , Polimorfismo Genético/fisiología , Reino Unido , Adulto JovenRESUMEN
CONTEXT: Three siblings of Pakistani origin presented neonatally with isolated hyperreninemic hypoaldosteronism and were well controlled on fludrocortisone therapy during childhood and adolescence. OBJECTIVE/DESIGN: These individuals were reevaluated as adults after fludrocortisone withdrawal to investigate the biochemical and molecular basis of their disorder. RESULTS: When reassessed off fludrocortisone treatment, hyperreninemic hypoaldosteronism was confirmed in all subjects but with significant hyperkalemia in only one case. Profiling of urinary steroid metabolites showed a biochemical pattern (elevated tetrahydrocorticosterone to 18-hydroxytetrahydro-11-dehydrocorticosterone ratio but normal 18-hydroxytetrahydro-11-dehydrocorticosterone to tetrahydroaldosterone ratio) consistent with partial type 1 aldosterone synthase deficiency. Sequencing of the CYP11B2 gene showed that affected subjects were homozygous for a single nucleotide substitution (T925C) in exon 5, corresponding to a serine to proline mutation (S308P) in the predicted protein, with unaffected family members being heterozygous. Consistent with structural modeling showing that the mutated residue is located within the alpha-helix I, close to the hemebinding, active site of the enzyme, functional characterization of the S308P mutant protein in vitro showed complete loss of enzyme activity. However, administration of dexamethasone further reduced levels of circulating aldosterone and its urinary metabolites in affected subjects, suggesting that some mineralocorticoid biosynthesis occurs in vivo. CONCLUSION: We have identified the first CYP11B2 gene defect in a family of Asian origin, associated with a type 1 aldosterone synthase deficiency phenotype. Preservation of some aldosterone production suggests either residual mutant CYP11B2 enzyme activity in vivo or mineralocorticoid biosynthesis via an alternative pathway.
Asunto(s)
Citocromo P-450 CYP11B2/deficiencia , Citocromo P-450 CYP11B2/genética , Mutación , Adulto , Aldosterona/sangre , Humanos , MasculinoRESUMEN
CONTEXT: Dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS) are the major circulating adrenal steroids and substrates for peripheral sex hormone biosynthesis. In Addison's disease, glucocorticoid and mineralocorticoid deficiencies require lifelong replacement, but the associated near-total failure of DHEA synthesis is not typically corrected. OBJECTIVE AND DESIGN: In a double-blind trial, we randomized 106 subjects (44 males, 62 females) with Addison's disease to receive either 50 mg daily of micronized DHEA or placebo orally for 12 months to evaluate its longer-term effects on bone mineral density, body composition, and cognitive function together with well-being and fatigue. RESULTS: Circulating DHEAS and androstenedione rose significantly in both sexes, with testosterone increasing to low normal levels only in females. DHEA reversed ongoing loss of bone mineral density at the femoral neck (P < 0.05) but not at other sites; DHEA enhanced total body (P = 0.02) and truncal (P = 0.017) lean mass significantly with no change in fat mass. At baseline, subscales of psychological well-being in questionnaires (Short Form-36, General Health Questionnaire-30), were significantly worse in Addison's patients vs. control populations (P < 0.001), and one subscale of SF-36 improved significantly (P = 0.004) after DHEA treatment. There was no significant benefit of DHEA treatment on fatigue or cognitive or sexual function. Supraphysiological DHEAS levels were achieved in some older females who experienced mild androgenic side effects. CONCLUSION: Although further long-term studies of DHEA therapy, with dosage adjustment, are desirable, our results support some beneficial effects of prolonged DHEA treatment in Addison's disease.
Asunto(s)
Enfermedad de Addison/tratamiento farmacológico , Deshidroepiandrosterona/uso terapéutico , Terapia de Reemplazo de Hormonas/métodos , Absorciometría de Fotón , Enfermedad de Addison/sangre , Adulto , Anciano , Composición Corporal/efectos de los fármacos , Densidad Ósea/efectos de los fármacos , Cognición/efectos de los fármacos , Sulfato de Deshidroepiandrosterona/sangre , Método Doble Ciego , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Calidad de Vida , Estadísticas no Paramétricas , Encuestas y CuestionariosRESUMEN
Extreme forms of insulin resistance are a rare cause of type 2 diabetes. However, individuals with severe insulin resistance pose unique diagnostic and therapeutic challenges, and have often acted as 'experiments of nature' providing important novel information regarding endocrine physiology and mechanistic insights relevant to the study of more common disorders. Progress in understanding the molecular pathogenesis of such syndromes is also beginning to yield novel therapeutic options. Severe insulin resistance typically presents in 1 of 3 ways: (1) disordered glucose metabolism including both diabetes and/or paradoxical hypoglycaemia; (2) acanthosis nigricans, a velvety hyperpigmentation of axilliary and flexural skin often associated with skin tags; or (3) hyperandrogenism in girls (hirsutism, oligo-/amenorrhoea and polycystic ovaries). Lipodystrophy is a major cause of severe insulin resistance and needs to be looked for very carefully, particularly in the patients with significant dyslipidaemia and fatty liver. Specific treatments are now available for some forms of severe insulin resistance; for example, leptin replacement in patients with generalized lipodystrophy. In the absence of a specific diagnosis and therapy, metformin is a useful insulin sensitizer and should be used in conjunction with aggressive diet and exercise interventions.
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Resistencia a la Insulina/fisiología , Síndrome Metabólico/terapia , Adolescente , Algoritmos , Femenino , Humanos , Síndrome Metabólico/clasificación , Síndrome Metabólico/diagnóstico , LinajeRESUMEN
The nuclear receptor family of PPARs was named for the ability of the original member to induce hepatic peroxisome proliferation in mice in response to xenobiotic stimuli. However, studies on the action and structure of the 3 human PPAR isotypes (PPARalpha, PPARdelta, and PPARgamma) suggest that these moieties are intimately involved in nutrient sensing and the regulation of carbohydrate and lipid metabolism. PPARalpha and PPARdelta appear primarily to stimulate oxidative lipid metabolism, while PPARgamma is principally involved in the cellular assimilation of lipids via anabolic pathways. Our understanding of the functions of PPARgamma in humans has been increased by the clinical use of potent agonists and by the discovery of both rare and severely deleterious dominant-negative mutations leading to a stereotyped syndrome of partial lipodystrophy and severe insulin resistance, as well as more common sequence variants with a much smaller impact on receptor function. These may nevertheless have much greater significance for the public health burden of metabolic disease. This Review will focus on the role of PPARgamma in human physiology, with specific reference to clinical pharmacological studies, and analysis of PPARG gene variants in the abnormal lipid and carbohydrate metabolism of the metabolic syndrome.
Asunto(s)
Enfermedades Metabólicas/fisiopatología , PPAR gamma/fisiología , Humanos , Enfermedades Metabólicas/genética , PPAR gamma/genéticaRESUMEN
The nuclear receptor superfamily comprises a group of proteins that includes the molecular targets for classical steroid hormones such as glucocorticoids, androgens and vitamin D, together with a number of so-called 'orphan' receptors whose ligands and/or function remain to be determined. Many of the world's most commonly prescribed drugs act via nuclear receptors, attesting to their importance as therapeutic targets in human disease [for example, the novel anti-diabetic thiazolidinediones rosiglitazone and pioglitazone are high-affinity ligands for peroxisome-proliferator-activated receptor gamma (PPARgamma)]. The study of transgenic mice harbouring global and tissue-specific alterations in nuclear receptor genes has greatly enhanced our understanding of the roles that these receptors play in mammalian physiology. In many cases, these findings have been complemented by the study of human subjects harbouring naturally occurring mutations within the corresponding receptor, whereas in others, such studies have served to highlight important differences that exist between human and mouse physiology especially, for example, in relation to aspects of metabolism. Here we review the diverse clinical phenotypes that have been reported in subjects found to have germline mutations in thyroid hormone receptor beta, PPARgamma, hepatocyte nuclear factor 4alpha, small heterodimer partner, steroidogenic factor 1, DAX1, photoreceptor-specific nuclear receptor and NUR-related factor 1, and consider the molecular mechanisms through which aberrant signalling by mutant receptors might contribute to the pathogenesis of the associated disorders.
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Enfermedad/etiología , Receptores Citoplasmáticos y Nucleares/genética , Animales , Mutación de Línea Germinal , Humanos , PPAR gamma/química , PPAR gamma/genética , PPAR gamma/fisiología , Fenotipo , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/fisiología , Receptores de Hormona Tiroidea/química , Receptores de Hormona Tiroidea/genética , Receptores de Hormona Tiroidea/fisiología , Factor Esteroidogénico 1 , Receptores beta de Hormona TiroideaRESUMEN
Subject- Peroxisome proliferator-activated receptor (PPAR)-gamma agonists are emerging as potential protectors against inflammatory cardiovascular diseases including atherosclerosis and diabetic complications. However, their molecular mechanism of action within vasculature remains unclear. We report here that PPARgamma agonists, thiazolidinedione class drugs (TZDs), or 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) were capable of activating diacylglycerol (DAG) kinase (DGK), resulting in attenuation of DAG levels and inhibition of protein kinase C (PKC) activation. The PPARgamma agonist-induced DGK was completely blocked by a dominant-negative mutant of PPARgamma, indicating an essential receptor-dependent action. Importantly, the suppression of DAG-PKC signaling pathway was functional linkage to the anti-inflammatory properties of PPARgamma agonists in endothelial cells (EC), characterized by the inhibition of proinflammatory adhesion molecule expression and adherence of monocytes to the activated EC induced by high glucose. These findings thus demonstrate a novel molecular action of PPARgamma agonists to suppress the DAG-PKC signaling pathway via upregulation of an endogenous attenuator, DGK.
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Cromanos/farmacología , Diacilglicerol Quinasa/fisiología , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , PPAR gamma/agonistas , Prostaglandina D2/análogos & derivados , Prostaglandina D2/farmacología , Tiazolidinedionas/farmacología , Animales , Aorta , Bovinos , Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/genética , Células Cultivadas/efectos de los fármacos , Angiopatías Diabéticas/metabolismo , Angiopatías Diabéticas/prevención & control , Diacilglicerol Quinasa/biosíntesis , Diacilglicerol Quinasa/genética , Diglicéridos/fisiología , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Inducción Enzimática/efectos de los fármacos , Glucosa/farmacología , Humanos , PPAR gamma/genética , Proteína Quinasa C/metabolismo , Proteína Quinasa C beta , Transporte de Proteínas/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/efectos de los fármacos , Transfección , Troglitazona , Células U937/citología , Células U937/efectos de los fármacos , Venas Umbilicales , Vasculitis/prevención & controlRESUMEN
Peroxisome proliferator-activated receptor-gamma (PPARgamma), an orphan nuclear receptor, mediates adipocyte differentiation and is the cellular target for the thiazolidinedione group of insulin-sensitizing antidiabetic agents. We screened this receptor gene in a cohort of subjects with severe insulin resistance and have identified heterozygous missense mutations in several individuals from three families. Functional studies indicate that the receptor mutants are transcriptionally impaired and inhibit wild type PPARgamma action in a dominant-negative manner. The clinical phenotype of patients includes partial lipodystrophy, early-onset hypertension, dyslipidaemia and hepatic steatosis. Factors which contribute to the severe insulin resistance in affected individuals include diminished body fat mass, impaired lipid flux in adipose tissue and reduced circulating levels of adiponectin. In a large kindred of five individuals with severe insulin resistance, we have identified frameshift/premature stop mutations in PPARGAMMA; and the muscle-specific regulatory subunit of protein phosphatase 1 (PPP1R3A). The frameshift PPARgamma mutant exhibits complete loss of function with no dominant-negative activity; the PPP1R3A truncation mutant is mislocalized intracellularly. Individuals harbouring either gene defect alone have normal circulating insulin levels, but a combination of both genetic abnormalities co-segregates with severe insulin resistance.
Asunto(s)
Resistencia a la Insulina/genética , Receptores Citoplasmáticos y Nucleares/genética , Factores de Transcripción/genética , Estudios de Cohortes , Mutación del Sistema de Lectura , Genes Dominantes , Humanos , Lipodistrofia/complicaciones , Lipodistrofia/genética , Lipodistrofia/metabolismo , Mutación Missense , Fenotipo , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismo , Proteína Fosfatasa 1 , Receptores Citoplasmáticos y Nucleares/fisiología , Factores de Transcripción/fisiologíaRESUMEN
Germline mutations in thyroid transcription factor 2 (TTF2) cause thyroid agenesis, spiky hair, and cleft palate, indicating thyroidal and extrathyroidal expression. We sought to investigate this by producing and applying an antibody to human TTF2. The coding region of human TTF2 was cloned into a bacterial expression vector, production of the soluble TTF2 protein optimized, and pure TTF2 obtained by nickel chromatography. Rabbits were immunized and the resulting TTF2 polyclonal titrated on formalin-fixed, paraffin-embedded sections of thyroid. The optimized protocol was applied to a range of tissues. Nine milligrams of TTF2 protein was obtained per liter of culture and a high-titer antibody produced. This displayed specific staining of thyroid follicular cell nuclei/cytoplasm and not of the interstitium, connective tissue, smooth muscle, or endothelium. No staining was obtained with the preimmune serum in the same conditions, or with the majority of other tissues tested with the TTF2 polyclonal. The exceptions were testis and skin, in which nuclear TTF2 immunoreactivity was present in the seminiferous tubules and cells in the follicular outer root sheath, respectively. In conclusion, we have produced a polyclonal antibody for human TTF2 and demonstrated immunoreactivity for this transcription factor in adult human thyroid and hair follicles and prepubertal testis.
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Proteínas de Unión al ADN/análisis , Proteínas Represoras/análisis , Testículo/fisiología , Glándula Tiroides/química , Adolescente , Adulto , Secuencia de Aminoácidos , Anticuerpos , Western Blotting , Niño , Proteínas de Unión al ADN/inmunología , Factores de Transcripción Forkhead , Folículo Piloso/química , Folículo Piloso/citología , Humanos , Inmunohistoquímica , Masculino , Datos de Secuencia Molecular , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/inmunología , Pubertad , Proteínas Represoras/inmunología , Testículo/citología , Glándula Tiroides/citologíaRESUMEN
By the end of this decade, it has been estimated that between 200 million and 300 million people worldwide will meet World Health Organization diagnostic criteria for diabetes mellitus. This epidemic of predominantly type 2 diabetes has largely been mediated by our shift toward a more sedentary lifestyle predisposing to obesity and insulin resistance. Affected individuals can also exhibit an array of associated undesirable traits such as hypertension, dyslipidemia, and hypercoagulability, leading to morbidity and mortality from atherosclerotic vascular disease. The coexistence of several of these traits with insulin resistance constitutes the metabolic syndrome. Accordingly, improving insulin sensitivity in this group, and thereby potentially ameliorating the excess vascular risk, is a primary goal of treatment. Recent interest has focused on the thiazolidinediones, a novel class of antidiabetic agents, which act as insulin sensitizers and, therefore, potentially target the underlying metabolic disturbance. These agents are high-affinity ligands for the nuclear receptor peroxisome proliferator-activated receptor gamma, and a large body of in vitro and in vivo data has evolved to support their increasing clinical use. Importantly, clinical and laboratory findings in human subjects harboring natural mutations and polymorphisms within the receptor have provided additional insights. Here, we focus on the consequences of inherited variation in the human peroxisome proliferator-activated receptor gamma gene, linking this receptor to disordered glucose homeostasis, adipogenesis, lipid metabolism, and blood pressure regulation. These studies provide further support for the future development of more selective receptor modulators, targeting specific pathways to ameliorate facets of the metabolic syndrome.
Asunto(s)
Hipoglucemiantes/uso terapéutico , Síndrome Metabólico/tratamiento farmacológico , Síndrome Metabólico/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Tiazoles/uso terapéutico , Tiazolidinedionas , Factores de Transcripción/metabolismo , Tejido Adiposo/crecimiento & desarrollo , Animales , Arteriosclerosis/genética , Humanos , Hiperlipidemias/genética , Hipertensión/genética , Resistencia a la Insulina , Mutación , Receptores Citoplasmáticos y Nucleares/genética , Factores de Transcripción/genéticaRESUMEN
We previously reported a syndrome of severe hyperinsulinemia and early-onset hypertension in three patients with dominant-negative mutations in the nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR)-gamma. We now report the results of further detailed pathophysiological evaluation of these subjects, the identification of affected prepubertal children within one of the original families, and the effects of thiazolidinedione therapy in two subjects. These studies 1) definitively demonstrate the presence of severe peripheral and hepatic insulin resistance in the affected subjects; 2) describe a stereotyped pattern of partial lipodystrophy associated with all the features of the metabolic syndrome and nonalcoholic steatohepatitis; 3) document abnormalities in the in vivo function of remaining adipose tissue, including the inability of subcutaneous abdominal adipose tissue to trap and store free fatty acids postprandially and the presence of very low circulating levels of adiponectin; 4) document the presence of severe hyperinsulinemia in prepubertal carriers of the proline-467-leucine (P467L) PPAR-gamma mutation; 5) provide the first direct evidence of cellular resistance to PPAR-gamma agonists in mononuclear cells derived from the patients; and 6) report on the metabolic response to thiazolidinedione therapy in two affected subjects. Although the condition is rare, the study of humans with dominant-negative mutations in PPAR-gamma can provide important insight into the roles of this nuclear receptor in human metabolism.
Asunto(s)
Síndrome Metabólico/genética , Mutación , Proteínas de Neoplasias , Receptores Citoplasmáticos y Nucleares/genética , Tiazolidinedionas , Factores de Transcripción/genética , Proteínas Supresoras de Tumor , Abdomen , Tejido Adiposo/metabolismo , Adulto , Composición Corporal , Proteínas Portadoras/genética , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Ácidos Grasos/metabolismo , Ácidos Grasos no Esterificados/metabolismo , Hígado Graso/genética , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Hiperlipidemias/genética , Resistencia a la Insulina/genética , Cinética , Hígado/efectos de los fármacos , Imagen por Resonancia Magnética , Masculino , Síndrome Metabólico/tratamiento farmacológico , Síndrome Metabólico/fisiopatología , Persona de Mediana Edad , Fenotipo , Receptores Citoplasmáticos y Nucleares/agonistas , Rosiglitazona , Tiazoles/uso terapéutico , Factores de Transcripción/agonistas , Triglicéridos/metabolismoRESUMEN
Nuclear receptors are transcription factors that activate gene expression in response to ligands. The C-terminal helix (helix 12) of the ligand-binding domain plays a critical role in the activation mechanism. When bound to activating ligands, helix 12 adopts a conformation that promotes the binding of co-activator proteins. Helix 12 also adopts this 'active' position in several ligand-free structures, raising questions as to the exact role of helix 12. We proposed that the dynamic properties of helix 12 may be critical for the activation mechanism and, to test this, have used fluorescence anisotropy techniques to directly monitor the mobility of helix 12 in PPARgamma. Our results suggest that helix 12 is significantly more mobile than the main body of the protein. Upon ligand binding, helix 12 shows reduced mobility, accounting for its role as a molecular switch. We also show that natural mutations in human PPARgamma, associated with severe insulin resistance and diabetes mellitus, exhibit perturbations in the dynamic behavior of helix 12. Our findings provide the first direct observations of the mobility of helix 12 and suggest that the dynamic properties of this helix are key to the regulation of transcriptional activity.
Asunto(s)
Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Sitios de Unión , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Polarización de Fluorescencia , Humanos , Técnicas In Vitro , Resistencia a la Insulina/genética , Resistencia a la Insulina/fisiología , Ligandos , Modelos Moleculares , Mutación , Conformación Proteica , Estructura Secundaria de Proteína , Receptores Citoplasmáticos y Nucleares/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factores de Transcripción/genéticaRESUMEN
Impaired insulin action is a key feature of type 2 diabetes and is also found, to a more extreme degree, in familial syndromes of insulin resistance. Although inherited susceptibility to insulin resistance may involve the interplay of several genetic loci, no clear examples of interactions among genes have yet been reported. Here we describe a family in which five individuals with severe insulin resistance, but no unaffected family members, were doubly [corrected] heterozygous with respect to frameshift/premature stop mutations in two unlinked genes, PPARG and PPP1R3A these encode peroxisome proliferator activated receptor gamma, which is highly expressed in adipocytes, and protein phosphatase 1, regulatory subunit 3, the muscle-specific regulatory subunit of protein phosphatase 1, which are centrally involved in the regulation of carbohydrate and lipid metabolism, respectively. That mutant molecules primarily involved in either carbohydrate or lipid metabolism can combine to produce a phenotype of extreme insulin resistance provides a model of interactions among genes that may underlie common human metabolic disorders such as type 2 diabetes.
Asunto(s)
Resistencia a la Insulina/genética , Fosfoproteínas Fosfatasas/genética , Receptores Citoplasmáticos y Nucleares/genética , Factores de Transcripción/genética , Adulto , Anciano , Animales , Células CHO , Cricetinae , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Diabetes Mellitus Tipo 2/genética , Femenino , Mutación del Sistema de Lectura , Heterocigoto , Humanos , Masculino , Persona de Mediana Edad , Linaje , Fosfoproteínas Fosfatasas/metabolismo , Proteína Fosfatasa 1 , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Ligands that specifically target retinoid-X receptors (RXRs) are emerging as potentially powerful therapies for cancer, diabetes, and the lowering of circulatory cholesterol. To date, RXR has only been crystallized in the absence of ligand or with the promiscuous ligand 9-cis retinoic acid, which also activates retinoic acid receptors. Here we present the structure of hRXRbeta in complex with the RXR-specific agonist LG100268 (LG268). The structure clearly reveals why LG268 is specific for the RXR ligand binding pocket and will not activate retinoic acid receptors. Intriguingly, in the crystals, the C-terminal "activation" helix (AF-2/helix H12) is trapped in a novel position not seen in other nuclear receptor structures such that it does not cap the ligand binding cavity. Mammalian two-hybrid assays indicate that LG268 is unable to release co-repressors from RXR unless co-activators are also present. Together these findings suggest that RXR ligands may be inefficient at repositioning helix H12.
Asunto(s)
Anticolesterolemiantes/farmacología , Ácidos Nicotínicos/farmacología , Receptores de Ácido Retinoico/agonistas , Tetrahidronaftalenos/farmacología , Factores de Transcripción/agonistas , Ligandos , Modelos Moleculares , Compuestos Orgánicos , Unión Proteica , Conformación Proteica , Receptores de Ácido Retinoico/química , Receptores de Ácido Retinoico/metabolismo , Receptores X Retinoide , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
To analyze the cardiovascular alterations thought to occur in resistance to thyroid hormone (RTH), cardiac involvement in 54 patients with RTH was investigated with the help of two-dimensional and Doppler echocardiography. Data from 41 of 54 adult subjects with RTH were also compared with those of 24 and 20 cases with hyperthyroidism (H) and hypothyroidism (h), respectively, as well as 22 healthy euthyroid controls (C). With respect to the type of mutations, no correlation was found between cardiovascular features and genotype. Compared with affected adults, children with RTH showed markedly higher serum free T3 (FT3), free T4 (FT4), and baseline TSH concentrations. Compared with healthy children of comparable age, RTH children had significantly higher heart rate and lower left ventricular (LV) ejection fraction (P = 0.006). Also, higher heart rate and FT4 as well as shorter diastolic relaxation of the myocardium (all P = 0.001) between RTH subjects with and without thyrotoxic cardiovascular features were found. Cardiac symptoms (palpitations, 32% vs. 71%) and signs (sinus tachycardia, 26% vs. 79%; atrial fibrillation, 6% vs. 17%) were significantly less frequent in RTH vs. H (all P = 0.001). Compared with C and h, heart rate, cardiac output, stroke volume, and systolic aortic flow velocity were strongly increased in RTH (all P = 0.0001) and H, although ejection (P = 0.0012) and shortening (P = 0.0001) fractions of the LV were markedly lower in RTH vs. H. Diastolic parameters, such as isovolumic relaxation (P = 0.0001) and deceleration time (P = 0.013), were shorter in RTH vs. h and C. In RTH, positive correlations between FT3 and heart rate, and between FT4 and LV ejection fraction were observed, whereas negative correlations between both FT3 and FT4 and isovolumic relaxation were noted. In conclusion, these findings indicate a modulated hyperthyroid effect on cardiac systolic and diastolic function of the myocardium in RTH, whereas other parameters, such as ejection and shortening fractions of the LV, systolic diameter, and LV wall thickness, were comparable to C. Differences in term of cardiovascular changes were smaller between the RTH and C groups than the RTH and the H or h groups. Thus, an incomplete cardiac response to thyroid hormone is present in RTH.