Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 35
Filtrar
Más filtros

Banco de datos
Tipo del documento
Intervalo de año de publicación
1.
Nature ; 567(7746): 43-48, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30760930

RESUMEN

Cell-identity switches, in which terminally differentiated cells are converted into different cell types when stressed, represent a widespread regenerative strategy in animals, yet they are poorly documented in mammals. In mice, some glucagon-producing pancreatic α-cells and somatostatin-producing δ-cells become insulin-expressing cells after the ablation of insulin-secreting ß-cells, thus promoting diabetes recovery. Whether human islets also display this plasticity, especially in diabetic conditions, remains unknown. Here we show that islet non-ß-cells, namely α-cells and pancreatic polypeptide (PPY)-producing γ-cells, obtained from deceased non-diabetic or diabetic human donors, can be lineage-traced and reprogrammed by the transcription factors PDX1 and MAFA to produce and secrete insulin in response to glucose. When transplanted into diabetic mice, converted human α-cells reverse diabetes and continue to produce insulin even after six months. Notably, insulin-producing α-cells maintain expression of α-cell markers, as seen by deep transcriptomic and proteomic characterization. These observations provide conceptual evidence and a molecular framework for a mechanistic understanding of in situ cell plasticity as a treatment for diabetes and other degenerative diseases.


Asunto(s)
Diabetes Mellitus/patología , Diabetes Mellitus/terapia , Células Secretoras de Glucagón/citología , Células Secretoras de Glucagón/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/patología , Animales , Biomarcadores/análisis , Linaje de la Célula/efectos de los fármacos , Reprogramación Celular/efectos de los fármacos , Diabetes Mellitus/inmunología , Diabetes Mellitus/metabolismo , Modelos Animales de Enfermedad , Femenino , Glucagón/metabolismo , Células Secretoras de Glucagón/efectos de los fármacos , Células Secretoras de Glucagón/trasplante , Glucosa/farmacología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/metabolismo , Factores de Transcripción Maf de Gran Tamaño/genética , Factores de Transcripción Maf de Gran Tamaño/metabolismo , Masculino , Ratones , Especificidad de Órganos/efectos de los fármacos , Polipéptido Pancreático/metabolismo , Células Secretoras de Polipéptido Pancreático/citología , Células Secretoras de Polipéptido Pancreático/efectos de los fármacos , Células Secretoras de Polipéptido Pancreático/metabolismo , Proteómica , Análisis de Secuencia de ARN , Transactivadores/genética , Transactivadores/metabolismo , Transcriptoma , Transducción Genética
2.
Nature ; 514(7523): 503-7, 2014 Oct 23.
Artículo en Inglés | MEDLINE | ID: mdl-25141178

RESUMEN

Total or near-total loss of insulin-producing ß-cells occurs in type 1 diabetes. Restoration of insulin production in type 1 diabetes is thus a major medical challenge. We previously observed in mice in which ß-cells are completely ablated that the pancreas reconstitutes new insulin-producing cells in the absence of autoimmunity. The process involves the contribution of islet non-ß-cells; specifically, glucagon-producing α-cells begin producing insulin by a process of reprogramming (transdifferentiation) without proliferation. Here we show the influence of age on ß-cell reconstitution from heterologous islet cells after near-total ß-cell loss in mice. We found that senescence does not alter α-cell plasticity: α-cells can reprogram to produce insulin from puberty through to adulthood, and also in aged individuals, even a long time after ß-cell loss. In contrast, before puberty there is no detectable α-cell conversion, although ß-cell reconstitution after injury is more efficient, always leading to diabetes recovery. This process occurs through a newly discovered mechanism: the spontaneous en masse reprogramming of somatostatin-producing δ-cells. The juveniles display 'somatostatin-to-insulin' δ-cell conversion, involving dedifferentiation, proliferation and re-expression of islet developmental regulators. This juvenile adaptability relies, at least in part, upon the combined action of FoxO1 and downstream effectors. Restoration of insulin producing-cells from non-ß-cell origins is thus enabled throughout life via δ- or α-cell spontaneous reprogramming. A landscape with multiple intra-islet cell interconversion events is emerging, offering new perspectives for therapy.


Asunto(s)
Envejecimiento/fisiología , Transdiferenciación Celular , Diabetes Mellitus Experimental/patología , Células Secretoras de Insulina/citología , Insulina/biosíntesis , Regeneración , Células Secretoras de Somatostatina/citología , Animales , Desdiferenciación Celular , Proliferación Celular , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 1/terapia , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/metabolismo , Células Secretoras de Glucagón/citología , Células Secretoras de Glucagón/metabolismo , Humanos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Ratones , Maduración Sexual , Somatostatina/biosíntesis , Somatostatina/metabolismo , Células Secretoras de Somatostatina/metabolismo
3.
Genes Dev ; 25(16): 1680-5, 2011 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-21852533

RESUMEN

Using single transcription factors to reprogram cells could produce important insights into the epigenetic mechanisms that direct normal differentiation, or counter inappropriate plasticity, or even provide new ways of manipulating normal ontogeny in vitro to control lineage diversification and differentiation. We enforced Pdx1 expression from the Neurogenin-3-expressing endocrine commitment point onward and found during the embryonic period a minor increased ß-cell allocation with accompanying reduced α-cell numbers. More surprisingly, almost all remaining Pdx1-containing glucagon/Arx-producing cells underwent a fairly rapid conversion at postnatal stages, through glucagon-insulin double positivity, to a state indistinguishable from normal ß cells, resulting in complete α-cell absence. This α-to-ß conversion was not caused by activating Pdx1 in the later glucagon-expressing state. Our findings reveal that Pdx1 can work single-handedly as a potent context-dependent autonomous reprogramming agent, and suggest a postnatal differentiation evaluation stage involved in normal endocrine maturation.


Asunto(s)
Células Secretoras de Glucagón/metabolismo , Proteínas de Homeodominio/metabolismo , Células Secretoras de Insulina/metabolismo , Transactivadores/metabolismo , Animales , Animales Recién Nacidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Glucagón/genética , Glucagón/metabolismo , Células Secretoras de Glucagón/citología , Proteínas de Homeodominio/genética , Inmunohistoquímica , Insulina/genética , Insulina/metabolismo , Células Secretoras de Insulina/citología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Páncreas/embriología , Páncreas/crecimiento & desarrollo , Páncreas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transactivadores/genética
4.
Nature ; 464(7292): 1149-54, 2010 Apr 22.
Artículo en Inglés | MEDLINE | ID: mdl-20364121

RESUMEN

Pancreatic insulin-producing beta-cells have a long lifespan, such that in healthy conditions they replicate little during a lifetime. Nevertheless, they show increased self-duplication after increased metabolic demand or after injury (that is, beta-cell loss). It is not known whether adult mammals can differentiate (regenerate) new beta-cells after extreme, total beta-cell loss, as in diabetes. This would indicate differentiation from precursors or another heterologous (non-beta-cell) source. Here we show beta-cell regeneration in a transgenic model of diphtheria-toxin-induced acute selective near-total beta-cell ablation. If given insulin, the mice survived and showed beta-cell mass augmentation with time. Lineage-tracing to label the glucagon-producing alpha-cells before beta-cell ablation tracked large fractions of regenerated beta-cells as deriving from alpha-cells, revealing a previously disregarded degree of pancreatic cell plasticity. Such inter-endocrine spontaneous adult cell conversion could be harnessed towards methods of producing beta-cells for diabetes therapies, either in differentiation settings in vitro or in induced regeneration.


Asunto(s)
Diferenciación Celular/fisiología , Transdiferenciación Celular/fisiología , Células Secretoras de Glucagón/citología , Células Secretoras de Insulina/citología , Animales , Biomarcadores/metabolismo , Recuento de Células , Muerte Celular/efectos de los fármacos , Linaje de la Célula , Proliferación Celular , Reprogramación Celular , Toxina Diftérica/farmacología , Toxina Diftérica/toxicidad , Femenino , Glucagón/biosíntesis , Glucagón/genética , Glucagón/metabolismo , Células Secretoras de Glucagón/metabolismo , Humanos , Insulina/biosíntesis , Insulina/metabolismo , Insulina/farmacología , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Masculino , Ratones , Ratones Transgénicos , Ratas , Regeneración/fisiología
5.
PLoS Genet ; 9(1): e1003274, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23382704

RESUMEN

All pancreatic endocrine cell types arise from a common endocrine precursor cell population, yet the molecular mechanisms that establish and maintain the unique gene expression programs of each endocrine cell lineage have remained largely elusive. Such knowledge would improve our ability to correctly program or reprogram cells to adopt specific endocrine fates. Here, we show that the transcription factor Nkx6.1 is both necessary and sufficient to specify insulin-producing beta cells. Heritable expression of Nkx6.1 in endocrine precursors of mice is sufficient to respecify non-beta endocrine precursors towards the beta cell lineage, while endocrine precursor- or beta cell-specific inactivation of Nkx6.1 converts beta cells to alternative endocrine lineages. Remaining insulin(+) cells in conditional Nkx6.1 mutants fail to express the beta cell transcription factors Pdx1 and MafA and ectopically express genes found in non-beta endocrine cells. By showing that Nkx6.1 binds to and represses the alpha cell determinant Arx, we identify Arx as a direct target of Nkx6.1. Moreover, we demonstrate that Nkx6.1 and the Arx activator Isl1 regulate Arx transcription antagonistically, thus establishing competition between Isl1 and Nkx6.1 as a critical mechanism for determining alpha versus beta cell identity. Our findings establish Nkx6.1 as a beta cell programming factor and demonstrate that repression of alternative lineage programs is a fundamental principle by which beta cells are specified and maintained. Given the lack of Nkx6.1 expression and aberrant activation of non-beta endocrine hormones in human embryonic stem cell (hESC)-derived insulin(+) cells, our study has significant implications for developing cell replacement therapies.


Asunto(s)
Células Endocrinas , Proteínas de Homeodominio , Células Secretoras de Insulina , Insulina , Animales , Diferenciación Celular/genética , Linaje de la Célula , Tratamiento Basado en Trasplante de Células y Tejidos , Células Endocrinas/citología , Células Endocrinas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Insulina/genética , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Factores de Transcripción Maf de Gran Tamaño/genética , Factores de Transcripción Maf de Gran Tamaño/metabolismo , Ratones , Páncreas/citología , Células Madre , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
6.
Diabetologia ; 58(2): 304-12, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25413047

RESUMEN

AIMS/HYPOTHESIS: Non-invasive imaging of beta cells is a much-needed development but is one that faces significant biological and technological hurdles. A relevant imaging method should at least allow for an evaluation over time of the mass of beta cells under physiological and pathological conditions, and for an assessment of novel therapies. We, therefore, investigated the ability of a new MRI probe to repeatedly measure the loss of beta cells in a rodent model. METHODS: We developed an innovative nanoparticle probe that targets the glucagon-like peptide 1 receptor, and can be used for both fluorescence imaging and MRI. Using fluorescence, we characterised the specificity and biodistribution of the probe. Using 1.5 T MRI, we longitudinally imaged the changes in insulin content in male and female mice of the RIP-DTr strain, which mimic the changes expected in type 1 and type 2 diabetes, respectively. RESULTS: We showed that this probe selectively labelled beta cells in situ, imaged in vivo native pancreatic islets and evaluated their loss after diphtheria toxin administration, in a model of graded beta cell deletion. Thus, using clinical MRI, the probe quantitatively differentiates, in the same mouse strain, between female animals featuring a 50% loss of beta cells and the males featuring an almost complete loss of beta cells. CONCLUSIONS/INTERPRETATION: The approach addresses several of the hurdles that have so far limited the non-invasive imaging of beta cells, including the potential to repeatedly monitor the very same animals using clinically available equipment, and to differentiate graded losses of beta cells.


Asunto(s)
Glucagón/metabolismo , Células Secretoras de Insulina/metabolismo , Imagen por Resonancia Magnética , Fragmentos de Péptidos/metabolismo , Receptores de Glucagón/metabolismo , Animales , Femenino , Masculino , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Sondas Moleculares , Distribución Tisular
7.
Acta Neuropathol Commun ; 10(1): 9, 2022 01 28.
Artículo en Inglés | MEDLINE | ID: mdl-35090564

RESUMEN

GNAO1 encephalopathy characterized by a wide spectrum of neurological deficiencies in pediatric patients originates from de novo heterozygous mutations in the gene encoding Gαo, the major neuronal G protein. Efficient treatments and even the proper understanding of the underlying etiology are currently lacking for this dominant disease. Adequate animal models of GNAO1 encephalopathy are urgently needed. Here we describe establishment and characterization of mouse models of the disease based on two point mutations in GNAO1 with different clinical manifestations. One of them is G203R leading to the early-onset epileptic seizures, motor dysfunction, developmental delay and intellectual disability. The other is C215Y producing much milder clinical outcomes, mostly-late-onset hyperkinetic movement disorder. The resultant mouse models show distinct phenotypes: severe neonatal lethality in GNAO1[G203R]/ + mice vs. normal vitality in GNAO1[C215Y]/ + . The latter model further revealed strong hyperactivity and hyperlocomotion in a panel of behavioral assays, without signs of epilepsy, recapitulating the patients' manifestations. Importantly, despite these differences the two models similarly revealed prenatal brain developmental anomalies, such as enlarged lateral ventricles and decreased numbers of neuronal precursor cells in the cortex. Thus, our work unveils GNAO1 encephalopathy as to a large extent neurodevelopmental malady. We expect that this understanding and the tools we established will be instrumental for future therapeutic developments.


Asunto(s)
Encefalopatías/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Trastornos del Neurodesarrollo/genética , Adolescente , Adulto , Anciano , Animales , Niño , Preescolar , Modelos Animales de Enfermedad , Epilepsia/genética , Femenino , Humanos , Masculino , Ratones , Mutación , Fenotipo
8.
Cell Rep ; 38(7): 110377, 2022 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-35172145

RESUMEN

The precise developmental dynamics of the pancreatic islet endocrine cell types, and their interrelation, are unknown. Some authors claim the persistence of islet cell differentiation from precursor cells after birth ("neogenesis"). Here, using four conditional cell lineage tracing ("pulse-and-chase") murine models, we describe the natural history of pancreatic islet cells, once they express a hormone gene, until late in life. Concerning the contribution of early-appearing embryonic hormone-expressing cells to the formation of islets, we report that adult islet cells emerge from embryonic hormone-expressing cells arising at different time points during development, without any evidence of postnatal neogenesis. We observe specific patterns of hormone gene activation and switching during islet morphogenesis, revealing that, within each cell type, cells have heterogeneous developmental trajectories. This likely applies to most maturating cells in the body, and explains the observed phenotypic variability within differentiated cell types. Such knowledge should help devising novel regenerative therapies.


Asunto(s)
Envejecimiento/fisiología , Feto/citología , Hormonas/metabolismo , Islotes Pancreáticos/citología , Islotes Pancreáticos/embriología , Animales , Doxiciclina/farmacología , Desarrollo Embrionario/efectos de los fármacos , Feto/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Glucagón/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Ratones Transgénicos , Somatostatina/metabolismo , Coloración y Etiquetado
9.
Nat Commun ; 13(1): 2020, 2022 04 19.
Artículo en Inglés | MEDLINE | ID: mdl-35440614

RESUMEN

Generation of surrogate cells with stable functional identities is crucial for developing cell-based therapies. Efforts to produce insulin-secreting replacement cells to treat diabetes require reliable tools to assess islet cellular identity. Here, we conduct a thorough single-cell transcriptomics meta-analysis to identify robustly expressed markers used to build genesets describing the identity of human α-, ß-, γ- and δ-cells. These genesets define islet cellular identities better than previously published genesets. We show their efficacy to outline cell identity changes and unravel some of their underlying genetic mechanisms, whether during embryonic pancreas development or in experimental setups aiming at developing glucose-responsive insulin-secreting cells, such as pluripotent stem-cell differentiation or in adult islet cell reprogramming protocols. These islet cell type-specific genesets represent valuable tools that accurately benchmark gain and loss in islet cell identity traits.


Asunto(s)
Células Secretoras de Insulina , Islotes Pancreáticos , Células Madre Pluripotentes , Diferenciación Celular/genética , Humanos , Insulina/genética
10.
Nat Commun ; 12(1): 7235, 2021 12 13.
Artículo en Inglés | MEDLINE | ID: mdl-34903763

RESUMEN

Developmental genes are frequently controlled by multiple enhancers sharing similar specificities. As a result, deletions of such regulatory elements have often failed to reveal their full function. Here, we use the Pitx1 testbed locus to characterize in detail the regulatory and cellular identity alterations following the deletion of one of its enhancers (Pen). By combining single cell transcriptomics and an in-embryo cell tracing approach, we observe an increased fraction of Pitx1 non/low-expressing cells and a decreased fraction of Pitx1 high-expressing cells. We find that the over-representation of Pitx1 non/low-expressing cells originates from a failure of the Pitx1 locus to coordinate enhancer activities and 3D chromatin changes. This locus mis-activation induces a localized heterochrony and a concurrent loss of irregular connective tissue, eventually leading to a clubfoot phenotype. This data suggests that, in some cases, redundant enhancers may be used to locally enforce a robust activation of their host regulatory landscapes.


Asunto(s)
Elementos de Facilitación Genéticos/genética , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción Paired Box/genética , Acetilación , Animales , Cromatina/química , Cromatina/metabolismo , Tejido Conectivo/crecimiento & desarrollo , Tejido Conectivo/metabolismo , Embrión de Mamíferos , Epigénesis Genética , Miembro Posterior/citología , Miembro Posterior/embriología , Miembro Posterior/metabolismo , Esbozos de los Miembros/citología , Esbozos de los Miembros/embriología , Esbozos de los Miembros/metabolismo , Ratones , Modelos Genéticos , Factores de Transcripción Paired Box/metabolismo , Eliminación de Secuencia
11.
Nat Commun ; 12(1): 4458, 2021 07 22.
Artículo en Inglés | MEDLINE | ID: mdl-34294685

RESUMEN

The cellular identity of pancreatic polypeptide (Ppy)-expressing γ-cells, one of the rarest pancreatic islet cell-type, remains elusive. Within islets, glucagon and somatostatin, released respectively from α- and δ-cells, modulate the secretion of insulin by ß-cells. Dysregulation of insulin production raises blood glucose levels, leading to diabetes onset. Here, we present the genetic signature of human and mouse γ-cells. Using different approaches, we identified a set of genes and pathways defining their functional identity. We found that the γ-cell population is heterogeneous, with subsets of cells producing another hormone in addition to Ppy. These bihormonal cells share identity markers typical of the other islet cell-types. In mice, Ppy gene inactivation or conditional γ-cell ablation did not alter glycemia nor body weight. Interestingly, upon ß-cell injury induction, γ-cells exhibited gene expression changes and some of them engaged insulin production, like α- and δ-cells. In conclusion, we provide a comprehensive characterization of γ-cells and highlight their plasticity and therapeutic potential.


Asunto(s)
Insulina/biosíntesis , Células Secretoras de Polipéptido Pancreático/metabolismo , Polipéptido Pancreático/metabolismo , Precursores de Proteínas/metabolismo , Animales , Glucemia/metabolismo , Peso Corporal , Linaje de la Célula/genética , Femenino , Técnicas de Sustitución del Gen , Humanos , Células Secretoras de Insulina/clasificación , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Masculino , Ratones , Ratones Transgénicos , Páncreas/citología , Páncreas/embriología , Páncreas/crecimiento & desarrollo , Polipéptido Pancreático/deficiencia , Polipéptido Pancreático/genética , Células Secretoras de Polipéptido Pancreático/clasificación , Células Secretoras de Polipéptido Pancreático/citología , Embarazo , RNA-Seq
12.
Gastroenterology ; 136(1): 309-319.e9, 2009 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-19022256

RESUMEN

BACKGROUND & AIMS: The pancreatic mass is determined by the coordinated expansion and differentiation of progenitor cells and is maintained via tight control of cell replacement rates. The basic helix-loop-helix transcription factor c-Myc is one of the main regulators of these processes in many organs. We studied the requirement of c-Myc in controlling the generation and maintenance of pancreatic mass. METHODS: We conditionally inactivated c-Myc in Pdx1+ pancreatic progenitor cells. Pancreata of mice lacking c-Myc (c-Myc(P-/-) mice) were analyzed during development and ageing. RESULTS: Pancreatic growth in c-Myc(P-/-) mice was impaired starting on E12.5, in early primordia, because of decreased proliferation and altered differentiation of exocrine progenitors; islet progenitors were spared. Acinar cell maturation was defective in the adult hypotrophic pancreas, which hampered exocrine mass maintenance in aged animals. From 2 to 10 months of age, the c-Myc(P-/-) pancreas was progressively remodeled without inflammatory injury. Loss of acinar cells increased with time, concomitantly with adipose tissue accumulation. Using a genetic cell lineage tracing analysis, we demonstrated that pancreatic adipose cells were derived directly from transdifferentiating acinar cells. This epithelial-to-mesenchyme transition was also observed in normal aged specimens and in pancreatitis. CONCLUSIONS: These results provide evidence indicating that c-Myc activity is required for growth and maturation of the exocrine pancreas, and sheds new light on the ontogeny of pancreatic adipose cells in processes of organ degenerescence and tissue involution.


Asunto(s)
Adipocitos/citología , Páncreas Exocrino/patología , Proteínas Proto-Oncogénicas c-myc/fisiología , Animales , Diferenciación Celular , Proliferación Celular , Epitelio/patología , Proteínas de Homeodominio/análisis , Humanos , Mesodermo/patología , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-myc/análisis , Transactivadores/análisis
13.
Nucleic Acids Res ; 35(9): 3053-63, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17452369

RESUMEN

FEN-1 and XPG are members of the FEN-1 family of structure-specific nucleases, which share a conserved active site. FEN-1 plays a central role in DNA replication, whereas XPG is involved in nucleotide excision repair (NER). Both FEN-1 and XPG are active on flap structures, but only XPG cleaves bubble substrates. The spacer region of XPG is dispensable for nuclease activity on flap substrates but is required for NER activity and for efficient processing of bubble substrates. Here, we inserted the spacer region of XPG between the nuclease domains of FEN-1 to test whether this domain would be sufficient to confer XPG-like substrate specificity and NER activity on a related nuclease. The resulting FEN-1-XPG hybrid protein is active on flap and, albeit at low levels, on bubble substrates. Like FEN-1, the activity of FEN-1-XPG was stimulated by a double-flap substrate containing a 1-nt 3' flap, whereas XPG does not show this substrate preference. Although no NER activity was detected in vitro, the FEN-1-XPG hybrid displays substantial NER activity in vivo. Hence, insertion of the XPG spacer region into FEN-1 results in a hybrid protein with biochemical properties reminiscent of both nucleases, including partial NER activity.


Asunto(s)
Reparación del ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Endonucleasas/química , Endonucleasas/metabolismo , Endonucleasas de ADN Solapado/química , Endonucleasas de ADN Solapado/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Células Cultivadas , Daño del ADN , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Endonucleasas de ADN Solapado/genética , Humanos , Proteínas Nucleares/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Especificidad por Sustrato , Factores de Transcripción/genética , Rayos Ultravioleta
14.
Cell Metab ; 29(3): 755-768.e5, 2019 03 05.
Artículo en Inglés | MEDLINE | ID: mdl-30713109

RESUMEN

Type 1 diabetes (T1D) results from the autoimmune destruction of insulin-producing ß cells. A comprehensive picture of the changes during T1D development is lacking due to limited sample availability, inability to sample longitudinally, and the paucity of technologies enabling comprehensive tissue profiling. Here, we analyzed 1,581 islets from 12 human donors, including eight with T1D, using imaging mass cytometry (IMC). IMC enabled simultaneous measurement of 35 biomarkers with single-cell and spatial resolution. We performed pseudotime analysis of islets through T1D progression from snapshot data to reconstruct the evolution of ß cell loss and insulitis. Our analyses revealed that ß cell destruction is preceded by a ß cell marker loss and by recruitment of cytotoxic and helper T cells. The approaches described herein demonstrate the value of IMC for improving our understanding of T1D pathogenesis, and our data lay the foundation for hypothesis generation and follow-on experiments.


Asunto(s)
Biomarcadores/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Citometría de Imagen/métodos , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Páncreas/metabolismo , Progresión de la Enfermedad , Humanos , Células Secretoras de Insulina/patología , Islotes Pancreáticos/patología , Páncreas/patología
16.
Nat Cell Biol ; 20(11): 1267-1277, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30361701

RESUMEN

The mechanisms that restrict regeneration and maintain cell identity following injury are poorly characterized in higher vertebrates. Following ß-cell loss, 1-2% of the glucagon-producing α-cells spontaneously engage in insulin production in mice. Here we explore the mechanisms inhibiting α-cell plasticity. We show that adaptive α-cell identity changes are constrained by intra-islet insulin- and Smoothened-mediated signalling, among others. The combination of ß-cell loss or insulin-signalling inhibition, with Smoothened inactivation in α- or δ-cells, stimulates insulin production in more α-cells. These findings suggest that the removal of constitutive 'brake signals' is crucial to neutralize the refractoriness to adaptive cell-fate changes. It appears that the maintenance of cell identity is an active process mediated by repressive signals, which are released by neighbouring cells and curb an intrinsic trend of differentiated cells to change.


Asunto(s)
Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Transducción de Señal , Receptor Smoothened/metabolismo , Animales , Diferenciación Celular , Plasticidad de la Célula , Proliferación Celular , Femenino , Células Secretoras de Glucagón/citología , Células Secretoras de Glucagón/metabolismo , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/citología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Ratones Transgénicos , Receptor Smoothened/genética
17.
Mol Cell Biol ; 24(24): 10670-80, 2004 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-15572672

RESUMEN

XPG is the human endonuclease that cuts 3' to DNA lesions during nucleotide excision repair. Missense mutations in XPG can lead to xeroderma pigmentosum (XP), whereas truncated or unstable XPG proteins cause Cockayne syndrome (CS), normally yielding life spans of <7 years. One XP-G individual who had advanced XP/CS symptoms at 28 years has been identified. The genetic, biochemical, and cellular defects in this remarkable case provide insight into the onset of XP and CS, and they reveal a previously unrecognized property of XPG. Both of this individual's XPG alleles produce a severely truncated protein, but an infrequent alternative splice generates an XPG protein lacking seven internal amino acids, which can account for his very slight cellular UV resistance. Deletion of XPG amino acids 225 to 231 does not abolish structure-specific endonuclease activity. Instead, this region is essential for interaction with TFIIH and for the stable recruitment of XPG to sites of local UV damage after the prior recruitment of TFIIH. These results define a new functional domain of XPG, and they demonstrate that recruitment of DNA repair proteins to sites of damage does not necessarily lead to productive repair reactions. This observation has potential implications that extend beyond nucleotide excision repair.


Asunto(s)
Daño del ADN/efectos de la radiación , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción TFII/metabolismo , Rayos Ultravioleta , Empalme Alternativo , Secuencia de Aminoácidos , Línea Celular , Línea Celular Transformada , Transformación Celular Viral , Análisis Mutacional de ADN , Reparación del ADN , Proteínas de Unión al ADN/genética , Endonucleasas/análisis , Endonucleasas/metabolismo , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Técnica del Anticuerpo Fluorescente Indirecta , Mutación del Sistema de Lectura , Humanos , Immunoblotting , Lentivirus/genética , Longevidad , Masculino , Microscopía Fluorescente , Proteínas Nucleares , Pruebas de Precipitina , Estructura Terciaria de Proteína , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Factor de Transcripción TFIIH , Factores de Transcripción , Xerodermia Pigmentosa/diagnóstico , Xerodermia Pigmentosa/genética , Xerodermia Pigmentosa/metabolismo , Xerodermia Pigmentosa/patología
18.
Int J Biochem Cell Biol ; 88: 226-235, 2017 07.
Artículo en Inglés | MEDLINE | ID: mdl-28119131

RESUMEN

In addition to ß-cells, pancreatic islets contain α- and δ-cells, which respectively produce glucagon and somatostatin. The reprogramming of these two endocrine cell types into insulin producers, as observed after a massive ß-cell ablation in mice, may help restoring a functional ß-cell mass in type 1 diabetes. Yet, the spontaneous α-to-ß and δ-to-ß conversion processes are relatively inefficient in adult animals and the underlying epigenetic mechanisms remain unclear. Several studies indicate that the conserved chromatin modifiers DNA methyltransferase 1 (Dnmt1) and Enhancer of zeste homolog 2 (Ezh2) are important for pancreas development and restrict islet cell plasticity. Here, to investigate the role of these two enzymes in α- and δ-cell development and fate maintenance, we genetically inactivated them in each of these two cell types. We found that loss of Dnmt1 does not enhance the conversion of α- or δ-cells toward a ß-like fate. In addition, while Dnmt1 was dispensable for the development of these two cell types, we noticed a gradual loss of α-, but not δ-cells in adult mice. Finally, we found that Ezh2 inactivation does not enhance α-cell plasticity, and, contrary to what is observed in ß-cells, does not impair α-cell proliferation. Our results indicate that both Dnmt1 and Ezh2 play distinct roles in the different islet cell types.


Asunto(s)
ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Células Secretoras de Glucagón/metabolismo , Homeostasis , Células Secretoras de Somatostatina/metabolismo , Animales , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/deficiencia , Proteína Potenciadora del Homólogo Zeste 2/deficiencia , Activación Enzimática , Células Secretoras de Glucagón/citología , Ratones , Células Secretoras de Somatostatina/citología
19.
Cell Metab ; 25(3): 622-634, 2017 03 07.
Artículo en Inglés | MEDLINE | ID: mdl-28215845

RESUMEN

Insulin-producing pancreatic ß cells in mice can slowly regenerate from glucagon-producing α cells in settings like ß cell loss, but the basis of this conversion is unknown. Moreover, it remains unclear if this intra-islet cell conversion is relevant to diseases like type 1 diabetes (T1D). We show that the α cell regulators Aristaless-related homeobox (Arx) and DNA methyltransferase 1 (Dnmt1) maintain α cell identity in mice. Within 3 months of Dnmt1 and Arx loss, lineage tracing and single-cell RNA sequencing revealed extensive α cell conversion into progeny resembling native ß cells. Physiological studies demonstrated that converted α cells acquire hallmark ß cell electrophysiology and show glucose-stimulated insulin secretion. In T1D patients, subsets of glucagon-expressing cells show loss of DNMT1 and ARX and produce insulin and other ß cell factors, suggesting that DNMT1 and ARX maintain α cell identity in humans. Our work reveals pathways regulated by Arx and Dnmt1 that are sufficient for achieving targeted generation of ß cells from adult pancreatic α cells.


Asunto(s)
Envejecimiento/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Células Secretoras de Glucagón/metabolismo , Proteínas de Homeodominio/metabolismo , Células Secretoras de Insulina/metabolismo , Factores de Transcripción/metabolismo , Adulto , Señalización del Calcio/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Niño , Preescolar , ADN (Citosina-5-)-Metiltransferasa 1 , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patología , Fenómenos Electrofisiológicos/efectos de los fármacos , Femenino , Eliminación de Gen , Regulación de la Expresión Génica/efectos de los fármacos , Glucagón/metabolismo , Células Secretoras de Glucagón/efectos de los fármacos , Glucosa/farmacología , Humanos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Masculino , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Adulto Joven
20.
Cell Rep ; 18(13): 3192-3203, 2017 03 28.
Artículo en Inglés | MEDLINE | ID: mdl-28355570

RESUMEN

Pancreatic α cells may process proglucagon not only to glucagon but also to glucagon-like peptide-1 (GLP-1). However, the biological relevance of paracrine GLP-1 for ß cell function remains unclear. We studied effects of locally derived insulin secretagogues on ß cell function and glucose homeostasis using mice with α cell ablation and with α cell-specific GLP-1 deficiency. Normally, intestinal GLP-1 compensates for the lack of α cell-derived GLP-1. However, upon aging and metabolic stress, glucose tolerance is impaired. This was partly rescued with the DPP-4 inhibitor sitagliptin, but not with glucagon administration. In isolated islets from these mice, glucose-stimulated insulin secretion was heavily impaired and exogenous GLP-1 or glucagon rescued insulin secretion. These data highlight the importance of α cell-derived GLP-1 for glucose homeostasis during metabolic stress and may impact on the clinical use of systemic GLP-1 agonists versus stabilizing local α cell-derived GLP-1 by DPP-4 inhibitors in type 2 diabetes.


Asunto(s)
Adaptación Fisiológica , Péptido 1 Similar al Glucagón/metabolismo , Células Secretoras de Glucagón/metabolismo , Glucagón/metabolismo , Glucosa/metabolismo , Homeostasis , Células Secretoras de Insulina/metabolismo , Envejecimiento/patología , Animales , Dieta Alta en Grasa , Toxina Diftérica/administración & dosificación , Toxina Diftérica/farmacología , Células Secretoras de Glucagón/efectos de los fármacos , Intolerancia a la Glucosa/complicaciones , Intolerancia a la Glucosa/patología , Prueba de Tolerancia a la Glucosa , Homeostasis/efectos de los fármacos , Humanos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/complicaciones , Obesidad/patología , Proproteína Convertasas/metabolismo , Ratas , Estrés Fisiológico/efectos de los fármacos
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA