RESUMEN
Context: Excessive body iron stores are a risk factor for decreased insulin sensitivity (SI) and diabetes. We hypothesized that transcriptional dysregulation of genes involved in iron metabolism in adipocytes causes insulin resistance. Objective and Design: To define the genetic regulation of iron metabolism and its role in SI, we used gene expression, genotype, and SI data from an African American cohort (N = 256). Replication studies were performed in independent European ancestry cohorts. In vitro studies in human adipocytes were performed to define the role of a selected gene in causing insulin resistance. Results: Among 62 transcripts representing iron homeostasis genes, expression of 30 in adipose tissue were correlated with SI. Transferrin (TF) and ferritin heavy polypeptide were most positively and negatively associated with SI, respectively. These observations were replicated in two independent European ancestry adipose data sets. The strongest cis-regulatory variant for TF expression (rs6785596; P = 7.84 × 10-18) was identified in adipose but not muscle or liver tissue. Variants significantly affected the normal relationship of serum ferritin to insulin resistance. Knockdown of TF in differentiated Simpson-Golabi-Behmel syndrome adipocytes by short hairpin RNA decreased intracellular iron, reduced maximal insulin-stimulated glucose uptake, and reduced Akt phosphorylation. Knockdown of TF caused differential expression of 465 genes, including genes involved in glucose transport, mitochondrial function, Wnt-pathway/ SI, chemokine activity, and obesity. Iron chelation recapitulated key changes in the expression profile induced by TF knockdown. Conclusion: Genetic regulation of TF expression in adipose tissue plays a novel role in regulating SI.
Asunto(s)
Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Resistencia a la Insulina/genética , Transferrina/genética , Tejido Adiposo/citología , Adulto , Negro o Afroamericano , Línea Celular , Estudios de Cohortes , Femenino , Ferritinas/sangre , Ferritinas/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/fisiología , Técnicas de Silenciamiento del Gen , Glucosa/metabolismo , Humanos , Insulina/metabolismo , Hierro/metabolismo , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , ARN Interferente Pequeño/metabolismo , Transferrina/metabolismo , Población Blanca , Adulto JovenAsunto(s)
Neoplasias Abdominales/patología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Disco Óptico/patología , Paraganglioma/patología , Policitemia/patología , Neovascularización Retiniana/diagnóstico , Somatostatinoma/patología , Neoplasias Abdominales/genética , Adolescente , Adulto , Niño , Análisis Mutacional de ADN , Eritropoyetina/sangre , Exudados y Transudados , Femenino , Fibrosis , Angiografía con Fluoresceína , Humanos , Masculino , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Paraganglioma/genética , Policitemia/genética , Neovascularización Retiniana/genética , Somatostatinoma/genéticaRESUMEN
Congenital polycythemias have diverse etiologies, including mutations in the hypoxia sensing pathway. These include HIF2A at exon 12, VHL gene (Chuvash polycythemia), and PHD2 mutations, which in one family was also associated with recurrent pheochromocytoma/paraganglioma (PHEO/PGL). Over the past two decades, we have studied seven unrelated patients with sporadic congenital polycythemia who subsequently developed PHEO/PGL with, until now, no discernible molecular basis. We now report a polycythemic patient with a novel germline HIF2A (F374Y) (exon 9) mutation, inherited from his mother, who developed PHEO/PGL. We show that this is a gain-of-function mutation and demonstrate no loss-of-heterozygosity or additional somatic mutation of HIF2A in the tumor, indicating HIF2A (F374Y) may be predisposing rather than causative of PHEO/PGL. This report, in view of two other concomitantly reported PHEO/PGL patients with somatic mutations of HIF2A and polycythemia, underscores the PHEO/PGL-promoting potential of mutations of HIF2A that alone are not sufficient for PHEO/PGL development.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Mutación de Línea Germinal , Paraganglioma/complicaciones , Paraganglioma/genética , Policitemia/complicaciones , Policitemia/genética , Anciano de 80 o más Años , Secuencia de Aminoácidos , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/química , Femenino , Humanos , Masculino , Persona de Mediana Edad , Modelos Moleculares , Datos de Secuencia Molecular , Policitemia/congénito , Conformación Proteica , Alineación de SecuenciaRESUMEN
We recently developed a cell culture system for hepatitis E virus (HEV) in PLC/PRF/5 cells, using a genotype 3 HEV (JE03-1760F strain). Thirteen generations of consecutive passages of culture supernatant were successfully carried out in PLC/PRF/5 cells, with the highest HEV load reaching 10(8) copies/ml in the culture medium. Based on continuous release of progenies into culture medium, 50% tissue culture infectivity doses were estimated to be 2.0 x 10(3) copies for wild-type JE03-1760F and 1.4 x 10(2) copies for p13 (progeny in the thirteenth passage). Earlier appearance and greater increase in the yield of progenies in the culture supernatant were evident in p13 compared with wild-type. The cell culture-produced variants in primary propagation (p0) and consecutive passages (p5 [fifth passage], p10 [tenth], and p13) differed from the wild-type virus by 1, 9, 18, and 19 nucleotides (nt), respectively, over the entire genome of 7226nt, excluding the poly(A) tail. Three of five non-synonymous mutations in p13 were shared by a variant (fifth passage) in another series of passages of JE03-1760F. These results suggest that adaptation of HEV variants to growth in vitro is associated with a limited number of mutations similar to hepatitis A virus.