RESUMEN
Hypothalamic regulation of feeding and energy expenditure is a fundamental and evolutionarily conserved neurophysiological process critical for survival. Dysregulation of these processes, due to environmental or genetic causes, can lead to a variety of pathological conditions ranging from obesity to anorexia. Melanocortins and endogenous cannabinoids (eCBs) have been implicated in the regulation of feeding and energy homeostasis; however, the interaction between these signaling systems is poorly understood. Here, we show that the eCB 2-arachidonoylglycerol (2-AG) regulates the activity of melanocortin 4 receptor (MC4R) cells in the paraventricular nucleus of the hypothalamus (PVNMC4R) via inhibition of afferent GABAergic drive. Furthermore, the tonicity of eCBs signaling is inversely proportional to energy state, and mice with impaired 2-AG synthesis within MC4R neurons weigh less, are hypophagic, exhibit increased energy expenditure, and are resistant to diet-induced obesity. These mice also exhibit MC4R agonist insensitivity, suggesting that the energy state-dependent, 2-AG-mediated suppression of GABA input modulates PVNMC4R neuron activity to effectively respond to the MC4R natural ligands to regulate energy homeostasis. Furthermore, post-developmental disruption of PVN 2-AG synthesis results in hypophagia and death. These findings illustrate a functional interaction at the cellular level between two fundamental regulators of energy homeostasis, the melanocortin and eCB signaling pathways in the hypothalamic feeding circuitry.
Asunto(s)
Cannabinoides/metabolismo , Metabolismo Energético/fisiología , Homeostasis/fisiología , Receptor de Melanocortina Tipo 4/fisiología , Animales , Ácidos Araquidónicos/fisiología , Peso Corporal , Endocannabinoides/fisiología , Ayuno , Conducta Alimentaria/fisiología , Prueba de Tolerancia a la Glucosa , Glicéridos/fisiología , Resistencia a la Insulina , Ratones , Obesidad/genética , Receptor de Melanocortina Tipo 4/agonistas , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Pseudohypoparathyroidism type 1A (PHP1A) is a genetic disorder caused by maternally inherited mutations in the gene Gnas. PHP1A is characterized by multiple hormone impairment, early onset obesity and cognitive impairment. Animal models of PHP1A are needed to investigate the mechanism of cognitive impairment. In the present study we used the cre-lox murine model to investigate behavior and cognition in maternally vs. paternally inherited gnas mutations expressed in the central nervous system. We observed a behavioral phenotype of decreased anxiety and impaired amygdala-based learning in the PHP1A mutant mouse model though there was no difference in hippocampal based learning tasks. In the elevated zero maze and open field analysis in the locomotor activity chambers, mutant mice showed diminished anxiety/increased impulsivity which could correlate with the attention deficit phenotype of children with PHP1A. The mutant mice also demonstrated poorer motor strength on the inverted screen test. These findings mirror some clinical features of PHP1A though overall the murine phenotype was milder than expected.
Asunto(s)
Amígdala del Cerebelo/fisiopatología , Conducta Animal/fisiología , Aprendizaje/fisiología , Seudohipoparatiroidismo/fisiopatología , Animales , Cromograninas/genética , Modelos Animales de Enfermedad , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Patrón de Herencia , Ratones , Ratones Endogámicos C57BL , Fenotipo , Seudohipoparatiroidismo/genéticaRESUMEN
BACKGROUND: Prader-Willi syndrome (PWS) is a complex neuroendocrine disorder affecting approximately 1/15,000-1/30,000 people. Unmet medical needs of individuals with PWS make it a rare disease that models the importance of multidisciplinary approaches to care with collaboration between academic centers, medical homes, industry, and parent organizations. Multidisciplinary clinics support comprehensive, patient-centered care for individuals with complex genetic disorders and their families. Value comes from improved communication and focuses on quality family-centered care. METHODS: Interviews with medical professionals, scientists, managed care experts, parents, and individuals with PWS were conducted from July 1 to December 1, 2016. Review of the literature was used to provide support. RESULTS: Data are presented based on consensus from these interviews by specialty focusing on unique aspects of care, research, and management. We have also defined the Center of Excellence beyond the multidisciplinary clinic. CONCLUSION: Establishment of clinics motivates collaboration to provide evidence-based new standards of care, increases the knowledge base including through randomized controlled trials, and offers an additional resource for the community. They have a role in global telemedicine, including to rural areas with few resources, and create opportunities for clinical work to inform basic and translational research. As a care team, we are currently charged with understanding the molecular basis of PWS beyond the known genetic cause; developing appropriate clinical outcome measures and biomarkers; bringing new therapies to change the natural history of disease; improving daily patient struggles, access to care, and caregiver burden; and decreasing healthcare load. Based on experience to date with a PWS multidisciplinary clinic, we propose a design for this approach and emphasize the development of "Centers of Excellence." We highlight the dearth of evidence for management approaches creating huge gaps in care practices as a means to illustrate the importance of the collaborative environment and translational approaches.
Asunto(s)
Manejo de la Enfermedad , Comunicación Interdisciplinaria , Síndrome de Prader-Willi/terapia , Conocimientos, Actitudes y Práctica en Salud , Personal de Salud/psicología , Humanos , Síndrome de Prader-Willi/diagnóstico , Encuestas y CuestionariosRESUMEN
OBJECTIVE: Hypothalamic obesity (HyOb) is a rare cause of rapid weight gain and early metabolic comorbidities. Effective treatment strategies are limited. The registry collected participant data and compared treatment approaches. METHODS: The International Registry of Hypothalamic Obesity Disorders (IRHOD) was created as a registry portal to provide education. Data collected from the initial 4 years were evaluated. RESULTS: Eighty-seven participants were included for analysis (median age: 27 years, range: 3-71 years). A total of 96.5% had obesity, and 3.5% had overweight at maximal weight. Seventy-five had brain tumors (86%)-the majority were craniopharyngiomas (72% of those with tumors). Nontumor etiologies included congenital brain malformation (4.6%), traumatic brain injury (3.4%), and genetic anomaly (2.3%). Ninety percent received obesity treatments including nutritional counseling (82%), pharmacotherapy (59%), bariatric surgery (8%), and vagal nerve stimulation (1%). Forty-six percent reported follow-up BMI results after obesity treatment. Surgery was most effective (median BMI decrease: -8.2 kg/m2 , median interval: 2.6 years), with lifestyle intervention (BMI: -3.4 kg/m2 , interval: 1.2 years) and pharmacological therapy (BMI: -2.3 kg/m2 , interval: 0.8 years) being less effective. Eighty percent of participants reporting follow-up weight remained in the obesity range. CONCLUSIONS: IRHOD identified a large cohort with self-reported HyOb. Surgical therapy was most effective at weight reduction. Nutritional counseling and pharmacotherapy modestly improved BMI. Stepwise treatment strategy for HyOb (including nutritional, pharmacological, and surgical therapies in an experienced center) may be most valuable.
Asunto(s)
Enfermedades Hipotalámicas/complicaciones , Obesidad/genética , Aumento de Peso/genética , Adolescente , Adulto , Anciano , Niño , Preescolar , Femenino , Humanos , Enfermedades Hipotalámicas/patología , Masculino , Persona de Mediana Edad , Sistema de Registros , Resultado del Tratamiento , Adulto JovenRESUMEN
BACKGROUND: Exposure of rodents to chronic high-fat diet (HFD) results in upregulation of inflammatory markers and proliferation of microglia within the mediobasal hypothalamus. Such hypothalamic inflammation is associated with metabolic dysfunction, central leptin resistance, and maintenance of obesity. Bariatric surgeries result in long-term stable weight loss and improved metabolic function. However, the effects of such surgical procedures on HFD-induced hypothalamic inflammation are unknown. We sought to characterize the effects of two bariatric surgical procedures, Roux-en-Y gastric bypass (RYGB) and biliary diversion (BD-IL), in female mice with particular emphasis on HFD-induced hypothalamic inflammation and microgliosis. METHODS: RYGB and BD-IL were performed on diet-induced obese (DIO) mice. Quantitative RT-PCR and fluorescent microscopy were used to evaluate hypothalamic inflammatory gene expression and microgliosis. Results were compared to lean (CD), DIO sham-surgerized mice (DIO-SHAM), and dietary weight loss (DIO-Rev) controls. RESULTS: In female mice, RYGB and BD-IL result in normalization of hypothalamic inflammatory gene expression and microgliosis within 8 weeks of surgery, despite ongoing exposure to HFD. Paralleling these results, the hypothalamic expression levels of the orexigenic neuropeptide Agrp and the anorexic response of surgical mice to exogenous leptin were comparable to lean controls (CD). In contrast, results from DIO-Rev mice were comparable to DIO-SHAM mice, despite transition back to standard rodent show and normalization of weight. CONCLUSION: Bariatric surgery attenuates HFD-induced hypothalamic inflammation and microgliosis and restores leptin sensitivity, despite ongoing exposure to HFD.
Asunto(s)
Cirugía Bariátrica , Hipotálamo/patología , Obesidad/cirugía , Animales , Dieta Alta en Grasa , Femenino , Inflamación/patología , Inflamación/cirugía , Ratones Endogámicos C57BLRESUMEN
Cell lineages of the early human gonad commit to one of the two mutually antagonistic organogenetic fates, the testis or the ovary. Some individuals with a 46,XX karyotype develop testes or ovotestes (testicular or ovotesticular disorder of sex development; TDSD/OTDSD), due to the presence of the testis-determining gene, SRY Other rare complex syndromic forms of TDSD/OTDSD are associated with mutations in pro-ovarian genes that repress testis development (e.g. WNT4); however, the genetic cause of the more common non-syndromic forms is unknown. Steroidogenic factor-1 (known as NR5A1) is a key regulator of reproductive development and function. Loss-of-function changes in NR5A1 in 46,XY individuals are associated with a spectrum of phenotypes in humans ranging from a lack of testis formation to male infertility. Mutations in NR5A1 in 46,XX women are associated with primary ovarian insufficiency, which includes a lack of ovary formation, primary and secondary amenorrhoea as well as early menopause. Here, we show that a specific recurrent heterozygous missense mutation (p.Arg92Trp) in the accessory DNA-binding region of NR5A1 is associated with variable degree of testis development in 46,XX children and adults from four unrelated families. Remarkably, in one family a sibling raised as a girl and carrying this NR5A1 mutation was found to have a 46,XY karyotype with partial testicular dysgenesis. These unique findings highlight how a specific variant in a developmental transcription factor can switch organ fate from the ovary to testis in mammals and represents the first missense mutation causing isolated, non-syndromic 46,XX testicular/ovotesticular DSD in humans.
Asunto(s)
Proteínas de Unión al ADN/genética , Trastorno del Desarrollo Sexual 46,XY/genética , Insuficiencia Ovárica Primaria/genética , Desarrollo Sexual/genética , Factor Esteroidogénico 1/genética , Adulto , Síndrome de Resistencia Androgénica/genética , Síndrome de Resistencia Androgénica/patología , Linaje de la Célula/genética , Niño , Trastorno del Desarrollo Sexual 46,XY/patología , Femenino , Gónadas/crecimiento & desarrollo , Gónadas/patología , Humanos , Cariotipo , Masculino , Mutación Missense , Ovario/crecimiento & desarrollo , Ovario/patología , Linaje , Insuficiencia Ovárica Primaria/patología , Procesos de Determinación del Sexo , Testículo/crecimiento & desarrollo , Testículo/patologíaRESUMEN
Arcuate AgRP neurons are critical for food intake. Two pathways leading to AgRP neuron activation and food intake include regulation by peripheral hormones leptin and ghrelin, and neuronal regulation via glutamatergic inputs. In a recent article in Cell Reports, Yang et al. demonstrate 'a third way,' regulation by resident astrocytes.
Asunto(s)
Proteína Relacionada con Agouti/metabolismo , Astrocitos/metabolismo , Neuronas/metabolismo , Receptor de Adenosina A1/metabolismo , AnimalesRESUMEN
Craniopharyngiomas (CP) are epithelial neoplasms generally found in the area of the pituitary and hypothalamus. Despite benign histology, these tumors and/or their treatment often result in significant, debilitating disorders of endocrine, neurological, behavioral, and metabolic systems. Severe obesity is observed in a high percentage of patients with CP resulting in significant comorbidities and negatively impacting quality of life. Obesity occurs as a result of hypothalamic damage and disruption of normal homeostatic mechanisms regulating energy balance. Such pathological weight gain, termed hypothalamic obesity (HyOb), is often severe and refractory to therapy. Unfortunately, neither lifestyle intervention nor pharmacotherapy has proven effective in the treatment of HyOb. Given the limited choices and poor results of these treatments, several groups have examined bariatric surgery as a treatment alternative for patients with CP-HyOb. While a large body of evidence exists supporting the use of bariatric surgery in the treatment of exogenous obesity and its comorbidities, its role in the treatment of HyOb has yet to be defined. To date, the existing literature on bariatric surgery in CP-HyOb is largely limited to case reports and series with short term follow-up. Here we review the current reports on the use of bariatric surgery in the treatment of CP-HyOb. We also compare these results to those reported for other populations of HyOb, including Prader-Willi Syndrome, Bardet-Biedl syndrome, and hypothalamic melanocortin signaling defects. While initial reports of bariatric surgery in CP-HyOb are promising, their limited scope makes it difficult to draw any substantial conclusions as to the long term safety and efficacy of bariatric surgery in CP-HyOb. There continues to be a need for more robust, controlled, prospective studies with long term follow-up in order to better define the role of bariatric surgery in the treatment of HyOb.
RESUMEN
Obesity, type 2 diabetes, and related diseases represent major health threats to modern society. Related pathophysiology of impaired neuronal function in hypothalamic control centers regulating metabolism and body weight has been dissected extensively and recent studies have started focusing on potential roles of astrocytes and microglia. The hypothalamic vascular system, however, which maintains the microenvironment necessary for appropriate neuronal function, has been largely understudied. We recently discovered that high fat/high sucrose diet exposure leads to increased hypothalamic presence of immunoglobulin G (IgG1). Investigating this phenomenon further, we have discovered a significant increase in blood vessel length and density in the arcuate nucleus (ARC) of the hypothalamus in mice fed a high fat/high sucrose diet, compared to matched controls fed standard chow diet. We also found a clearly increased presence of α-smooth muscle actin immunoreactive vessels, which are rarely present in the ARC and indicate an increase in the formation of new arterial vessels. Along the blood brain barrier, an increase of degenerated endothelial cells are observed. Moreover, such hypothalamic angiogenesis was not limited to rodent models. We also found an increase in the number of arterioles of the infundibular nucleus (the human equivalent of the mouse ARC) in patients with type 2 diabetes, suggesting angiogenesis occurs in the human hypothalamus of diabetics. Our discovery reveals novel hypothalamic pathophysiology, which is reminiscent of diabetic retinopathy and suggests a potential functional involvement of the hypothalamic vasculature in the later stage pathogenesis of metabolic syndrome.
RESUMEN
Proper cell fate determination in mammalian gonads is critical for the establishment of sexual identity. The Hedgehog (Hh) pathway has been implicated in cell fate decision for various organs, including gonads. Desert Hedgehog (Dhh), one of the three mammalian Hh genes, has been implicated with other genes in the establishment of mouse fetal Leydig cells. To investigate whether Hh alone is sufficient to induce fetal Leydig cell differentiation, we ectopically activated the Hh pathway in Steroidogenic factor 1 (SF1)-positive somatic cell precursors of fetal ovaries. Hh activation transformed SF1-positive somatic ovarian cells into functional fetal Leydig cells. These ectopic fetal Leydig cells produced androgens and insulin-like growth factor 3 (INLS3) that cause virilization of female embryos and ovarian descent. However, the female reproductive system remained intact, indicating a typical example of female pseudohermaphroditism. The appearance of fetal Leydig cells was a direct consequence of Hh activation as evident by the absence of other testicular components in the affected ovary. This study provides not only insights into mechanisms of cell lineage specification in gonads, but also a model to understand defects in sexual differentiation.
Asunto(s)
Trastornos del Desarrollo Sexual/metabolismo , Feto , Proteínas Hedgehog/metabolismo , Células Intersticiales del Testículo/metabolismo , Ovario/metabolismo , Animales , Diferenciación Celular/genética , Trastornos del Desarrollo Sexual/genética , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Femenino , Feto/citología , Feto/metabolismo , Proteínas Hedgehog/genética , Inmunohistoquímica , Hibridación in Situ , Células Intersticiales del Testículo/citología , Células Intersticiales del Testículo/fisiología , Masculino , Ratones , Ratones Transgénicos , Diferenciación Sexual , Transducción de Señal/genéticaRESUMEN
Sexually dimorphic development of the gonads is controlled by positive and negative regulators produced by somatic cells. Many Wnt ligands, including ones that signal via the canonical beta-catenin pathway, are expressed in fetal gonads. beta-catenin, a key transcriptional regulator of the canonical Wnt pathway and an element of the cell adhesion complex, is essential for various aspects of embryogenesis. To study the involvement of beta-catenin in sex determination, we ablated beta-catenin specifically in the SF1-positive population of somatic cells. Although beta-catenin was present in gonads of both sexes, it was necessary only for ovarian differentiation but dispensable for testis development. Loss of beta-catenin in fetal testes did not affect Sertoli cell differentiation, testis morphogenesis or masculinization of the embryos. However, we observed molecular and morphological defects in ovaries lacking beta-catenin, including formation of testis-specific coelomic vessel, appearance of androgen-producing adrenal-like cells and loss of female germ cells. These phenotypes were strikingly similar to those found in the R-spondin1 (Rspo1) and Wnt4 knockout ovaries. In the absence of beta-catenin, expression of Wnt4 was down-regulated while that of Rspo1 was not affected, placing beta-catenin as a component in between Rspo1 and Wnt4. Our results demonstrate that beta-catenin is responsible for transducing sex-specific signals in the SF1-positive somatic cell population during mouse gonadal development.
Asunto(s)
Gónadas/crecimiento & desarrollo , Diferenciación Sexual , beta Catenina/metabolismo , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Gónadas/embriología , Gónadas/metabolismo , Masculino , Ratones , Ratones Transgénicos , Factores de Empalme de ARN , Transducción de Señal , Especificidad de la Especie , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , beta Catenina/genéticaRESUMEN
The nuclear receptor steroidogenic factor 1 (Sf1, Nr5a1) is essential for adrenal development and regulates genes that specify differentiated adrenocortical function. The transcriptional coactivator beta-catenin reportedly synergizes with Sf1 to regulate a subset of these target genes; moreover, Wnt family members, signaling via beta-catenin, are also implicated in adrenocortical development. To investigate the role of beta-catenin in the adrenal cortex, we used two Sf1/Cre transgenes to inactivate conditional beta-catenin alleles. Inactivation of beta-catenin mediated by Sf1/Cre(high), a transgene expressed at high levels, caused adrenal aplasia in newborn mice. Analysis of fetal adrenal development with Sf1/Cre(high)-mediated beta-catenin inactivation showed decreased proliferation in presumptive adrenocortical precursor cells. By contrast, the Sf1/Cre(low) transgene effected a lesser degree of beta-catenin inactivation that did not affect all adrenocortical cells, permitting adrenal survival to reveal age-dependent degeneration of the cortex. These results define crucial roles for beta-catenin--presumably as part of the Wnt canonical signaling pathway--in both embryonic development of the adrenal cortex and in maintenance of the adult organ.
Asunto(s)
Corteza Suprarrenal/metabolismo , Factor Esteroidogénico 1/metabolismo , beta Catenina/metabolismo , Corteza Suprarrenal/citología , Corteza Suprarrenal/embriología , Corteza Suprarrenal/crecimiento & desarrollo , Animales , Biomarcadores , Diferenciación Celular , Proliferación Celular , Regulación del Desarrollo de la Expresión Génica , Integrasas/genética , Integrasas/metabolismo , Ratones , Ratones Noqueados , Transducción de Señal , Factor Esteroidogénico 1/genética , Proteínas Wnt/metabolismo , beta Catenina/deficiencia , beta Catenina/genéticaRESUMEN
Leptin, an adipocyte-derived hormone, has emerged as a critical regulator of energy homeostasis. The leptin receptor (Lepr) is expressed in discrete regions of the brain; among the sites of highest expression are several mediobasal hypothalamic nuclei known to play a role in energy homeostasis, including the arcuate nucleus, the ventromedial hypothalamic nucleus (VMH), and the dorsomedial hypothalamic nucleus. Although most studies have focused on leptin's actions in the arcuate nucleus, the role of Lepr in these other sites has received less attention. To explore the role of leptin signaling in the VMH, we used bacterial artificial chromosome transgenesis to target Cre recombinase to VMH neurons expressing steroidogenic factor 1, thereby inactivating a conditional Lepr allele specifically in steroidogenic factor 1 neurons of the VMH. These knockout (KO) mice, designated Lepr KO(VMH), exhibited obesity, particularly when challenged with a high-fat diet. On a low-fat diet, Lepr KO(VMH) mice exhibited significantly increased adipose mass even when their weights were comparable to wild-type littermates. Furthermore, these mice exhibited a metabolic syndrome including hepatic steatosis, dyslipidemia, and hyperleptinemia. Lepr KO(VMH) mice were hyperinsulinemic from the age of weaning and eventually developed overt glucose intolerance. These data define nonredundant roles of the Lepr in VMH neurons in energy homeostasis and provide a model system for studying other actions of leptin in the VMH.
Asunto(s)
Adiposidad/genética , Síndrome Metabólico/genética , Receptores de Leptina/genética , Núcleo Hipotalámico Ventromedial/metabolismo , Animales , Composición Corporal/genética , Peso Corporal/genética , Ingestión de Alimentos/genética , Sistema Endocrino/fisiología , Metabolismo Energético/genética , Femenino , Glucosa/metabolismo , Homeostasis/genética , Integrasas/genética , Masculino , Ratones , Ratones Noqueados , Neuronas/metabolismo , Especificidad de Órganos , Receptores de Leptina/metabolismoRESUMEN
Targeted mutagenesis of Fgf9 in mice causes male-to-female sex reversal. Among the four FGF receptors, FGFR2 showed two highly specific patterns based on antibody staining, suggesting that it might be the receptor-mediating FGF9 signaling in the gonad. FGFR2 was detected at the plasma membrane in proliferating coelomic epithelial cells and in the nucleus in Sertoli progenitor cells. This expression pattern suggested that Fgfr2 might play more than one role in testis development. To test the hypothesis that Fgfr2 is required for male sex determination, we crossed mice carrying a floxed allele of Fgfr2 with two different Cre lines to induce a temporal or cell-specific deletion of this receptor. Results show that deletion of Fgfr2 in embryonic gonads phenocopies deletion of Fgf9 and leads to male-to-female sex reversal. Using these two Cre lines, we provide the first genetic evidence that Fgfr2 plays distinct roles in proliferation and Sertoli cell differentiation during testis development.
Asunto(s)
Diferenciación Celular/genética , Morfogénesis/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/fisiología , Células de Sertoli/citología , Procesos de Determinación del Sexo , Testículo/embriología , Animales , Proliferación Celular , Eliminación de Gen , Expresión Génica , Proteínas del Grupo de Alta Movilidad/genética , Integrasas/genética , Masculino , Ratones , Ratones Transgénicos , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Factor de Transcripción SOX9 , Testículo/citología , Testículo/metabolismo , Factores de Transcripción/genéticaRESUMEN
OBJECTIVE: Steroidogenic factor 1 (SF1), officially designated NR5A1, is a nuclear receptor that plays key roles in endocrine development and function. Previous reports of human SF1 mutations revealed a spectrum of phenotypes affecting adrenal function and/or gonadal development and sex differentiation. We present the clinical phenotype and functional effects of a novel SF1 mutation. PATIENT: The patient is a 22-year-old 46, XY Japanese patient who presented with dysgenetic testes, atrophic vasa deferentia and epididymides, lack of Müllerian structures, and clitoromegaly. Endocrine studies revealed normal adrenal function. RESULTS: Analysis of the SF1 gene revealed compound heterozygosity for a previously described p.G146A polymorphism and a novel missense mutation (p.R84C) in the accessory DNA-binding domain. The father carried the p.G146A polymorphism and the mother had the p.R84C mutation; both were clinically and reproductively normal. Functional studies demonstrated that the p.R84C SF1 had normal nuclear localization but decreased DNA-binding affinity and transcriptional activity compared with wild-type SF1; it did not exhibit any dominant negative activity. CONCLUSIONS: These results describe the human phenotype that results from compound heterozygosity of the p.G146A polymorphism and a novel p.R84C mutation of SF1, thereby extending the spectrum of human SF1 mutations that impair testis development and sex differentiation in a sex-limited manner while preserving normal adrenal function.
Asunto(s)
Proteínas de Unión al ADN/genética , Disgenesia Gonadal 46 XY/genética , Proteínas de Homeodominio/genética , Receptores Citoplasmáticos y Nucleares/genética , Factores de Transcripción/genética , Insuficiencia Suprarrenal/genética , Insuficiencia Suprarrenal/fisiopatología , Adulto , Células Cultivadas , Análisis Mutacional de ADN , Proteínas de Unión al ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Femenino , Técnica del Anticuerpo Fluorescente , Disgenesia Gonadal 46 XY/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Luciferasas/genética , Mutación/fisiología , Plásmidos/genética , Polimorfismo Genético/genética , Polimorfismo Genético/fisiología , Receptores Citoplasmáticos y Nucleares/metabolismo , Factor Esteroidogénico 1 , Receptores alfa de Hormona Tiroidea/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
The Cre-loxP strategy provides an approach to disrupt genes in specific tissues and/or cell types, circumventing lethality associated with global knockouts or secondary effects due to gene inactivation at other sites. A critical component is the development of transgenes that target Cre expression to specific cell types. Here, we describe the use of bacterial artificial chromosome (BAC) transgenesis to target Cre expression to tissues that express steroidogenic factor 1 (SF-1, officially designated Nr5a1). Consistent with the SF-1 expression pattern, the SF-1 BAC directed Cre expression to the somatic cells of the gonads, the adrenal cortex, the anterior pituitary, the spleen, and the ventromedial hypothalamic nucleus. This transgene provides a powerful tool to inactivate genes of interest in these tissues.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Integrasas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismo , Corteza Suprarrenal/metabolismo , Animales , Cromosomas Artificiales Bacterianos , Embrión de Mamíferos/citología , Embrión de Mamíferos/enzimología , Femenino , Marcación de Gen , Proteínas de Homeodominio/genética , Integrasas/genética , Masculino , Ratones , Ratones Transgénicos , Especificidad de Órganos , Ovario/metabolismo , Adenohipófisis/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Bazo/metabolismo , Factor Esteroidogénico 1 , Testículo/metabolismo , Distribución Tisular , Factores de Transcripción/genética , Transgenes , Núcleo Hipotalámico Ventromedial/metabolismoRESUMEN
The orphan nuclear receptor steroidogenic factor 1 (SF-1) regulates the differentiation and function of endocrine glands. Although SF-1 is constitutively active in cell-based assays, it is not known whether this transcriptional activity is modulated by ligands. Here, we describe the 1.5 angstroms crystal structure of the SF-1 ligand binding domain in complex with an LXXLL motif from a coregulator protein. The structure reveals the presence of a phospholipid ligand in a surprisingly large pocket (approximately 1600 angstroms3), with the receptor adopting the canonical active conformation. The bound phospholipid is readily exchanged and modulates SF-1 interactions with coactivators. Mutations designed to reduce the size of the SF-1 pocket or to disrupt hydrogen bonds with the phospholipid abolish SF-1/coactivator interactions and significantly reduce SF-1 transcriptional activity. These findings provide evidence that SF-1 is regulated by endogenous ligands and suggest an unexpected relationship between phospholipids and endocrine development and function.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Fosfolípidos/metabolismo , Conformación Proteica , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cristalografía , Proteínas de Unión al ADN/química , Histona Acetiltransferasas , Proteínas de Homeodominio , Enlace de Hidrógeno , Ligandos , Ratones , Datos de Secuencia Molecular , Mutación/genética , Coactivador 1 de Receptor Nuclear , Receptores Citoplasmáticos y Nucleares , Homología de Secuencia de Aminoácido , Factor Esteroidogénico 1 , Factores de Transcripción/química , Transcripción GenéticaRESUMEN
Steroidogenic factor 1 (SF-1) is an orphan nuclear receptor that plays key roles in endocrine development and function. Knockout mice lacking SF-1 have adrenal and gonadal agenesis, impaired gonadotropin expression, and structural abnormalities of the ventromedial hypothalamic nucleus. Previous studies have identified three human subjects with mutations in SF-1 causing adrenocortical insufficiency with varying degrees of gonadal dysfunction. We now describe a novel 8-bp microdeletion of SF-1, isolated from a 46, XY patient who presented with gonadal agenesis but normal adrenal function, which causes premature termination upstream of sequences encoding the activation function 2 domain. In cell transfection experiments, the mutated protein possessed no intrinsic transcriptional activity but rather inhibited the function of the wild-type protein in most cell types. To our knowledge, this is the first example of an apparent dominant-negative effect of a SF-1 mutation in humans. These findings, which define a SF-1 mutation that apparently differentially affects its transcriptional activity in vivo in the adrenal cortex and the gonads, may be relevant to the cohort of patients who present with 46, XY sex reversal but normal adrenal function.
Asunto(s)
Glándulas Suprarrenales/fisiopatología , Proteínas de Unión al ADN/genética , Trastornos del Desarrollo Sexual , Eliminación de Gen , Factores de Transcripción/genética , Adulto , Secuencia de Bases , Femenino , Factores de Transcripción Fushi Tarazu , Genes Dominantes , Genitales Femeninos/anomalías , Proteínas de Homeodominio , Humanos , Ligandos , Mutación , Receptores Citoplasmáticos y Nucleares , Factor Esteroidogénico 1 , Transcripción GenéticaRESUMEN
Gramicidin A (gA) is prototypical peptide antibiotic and a model ion channel former. Configured in the solid-state NMR beta(6.5)-helix channel conformation, gA was subjected to 1-ns molecular dynamics (MD) gas phase simulations using the all-atom charmm22 force field to ascertain the conformational stability of the Trp side chains as governed by backbone and neighboring side-chain contacts. Three microcanonical trajectories were computed using different initial atomic velocities for each of twenty different initial structures. For each set, one of the four Trp side chains in each monomer was initially positioned in one of the five non-native conformations (A. E. Dorigo et al., Biophysical Journal, 1999, Vol. 76, 1897-1908), the other Trps being positioned in the native state, o1. In three additional control simulations, all Trps were initiated in the native conformation. After equilibration, constraints were removed and subsequent conformational changes of the initially constrained Trp were measured. The chi(1) was more flexible than chi(2.1). The energetically optimal orientation, o1 (Dorigo et al., 1999), was the most stable in all four Trp positions (9, 11, 13, 15) and remained unchanged for the entire 1 ns simulation in 19 of 24 trials. Changes in chi(1) from each of the 5 suboptimal states occur readily. Two of the non-native conformations reverted readily to o1, whereas the other three converted to an intermediate state, i2. There were frequent interconversions between i2 and o1. We speculate that experimentally observed Trp stability is caused by interactions with the lipid-water interface, and that stabilization of one of the suboptimal conformations in gA, such as i2, by lipid headgroups could produce a secondary, metastable conformational state. This could explain recent experimental studies of differences in the channel conductance dispersity between gA and a Trp-to-Phe gA analog, gramicidin M (gM, J. C. Markham et al., Biochimica et Biophysica Acta, 2001, Vol. 1513, 185-192).