RESUMEN
Prospects for inducing endogenous beta-cell regeneration in the pancreas, one of the most attractive approaches to reverse type 1 and type 2 diabetes, have gained substantially from recent evidence that cells in the adult pancreas exhibit more plasticity than previously recognized. There are two major pathways to beta-cell regeneration, beta-cell replication and beta-cell neogenesis. Substantial evidence for a role for both processes exists in different models. While beta-cell replication clearly occurs during development and early in life, the potential for replication appears to decline substantially with age. In contrast, we have demonstrated that the exocrine compartment of the adult human pancreas contains a facultative stem cell that can differentiate into beta-cells under specific circumstances. We have favoured the idea that, similar to models described in liver regeneration, beta-cell mass can be increased either by neogenesis or replication, depending on the intensity of different stimuli or stressors. Understanding the nature of endocrine stem/progenitor cells and the mechanism by which external stimuli mobilize them to exhibit endocrine differentiation is central for success in therapeutic approaches to induce meaningful endogenous beta-cell neogenesis.
Asunto(s)
Diferenciación Celular/fisiología , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 2/patología , Células Secretoras de Insulina/fisiología , Páncreas/patología , Regeneración/fisiología , Adulto , Proliferación Celular , Células Cultivadas , Humanos , Células Secretoras de Insulina/citología , Masculino , Páncreas/citologíaRESUMEN
The basic helix-loop-helix transcription factor NeuroD1 regulates cell fate in the nervous system but previously has not been considered to function similarly in the endocrine pancreas due to its reported expression in all islet cell types in the newborn mouse. Because we found that NeuroD1 potently represses somatostatin expression in vitro, its pattern of expression was examined in both strains of mice in which lacZ has been introduced into the NeuroD1 locus by homologous recombination. Analysis of adult transgenic mice revealed that NeuroD1 is predominantly expressed in beta-cells and either absent or expressed below the limit of lacZ detection in mature alpha-, delta-, or PP cells. Consistent with a previous report, NeuroD1 colocalizes with glucagon as well as insulin in immature islets of the newborn mouse. However, no colocalization of NeuroD1with somatostatin was detected in the newborn. In vitro, ectopic expression of NeuroD1 in TRM-6/PDX-1, a human pancreatic delta-cell line, resulted in potent repression of somatostatin concomitant with induction of the beta-cell hormones insulin and islet amyloid polypeptide. Additionally, NeuroD1 induced expression of Nkx2.2, a transcription factor expressed in beta- but not delta-cells. Transfection studies using insulin and somatostatin promoters confirm the ability of NeuroD1 to act as both a transcriptional repressor and activator in the same cell, suggesting a more complex role for NeuroD1 in the establishment and/or maintenance of mature endocrine cells than has been recognized previously.
Asunto(s)
Perfilación de la Expresión Génica , Proteínas del Tejido Nervioso/metabolismo , Páncreas/metabolismo , Proteínas Represoras/metabolismo , Transactivadores/metabolismo , Envejecimiento/fisiología , Amiloide/metabolismo , Animales , Animales Recién Nacidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Línea Celular , ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Insulina/genética , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Ratones , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares , Páncreas/citología , Páncreas/crecimiento & desarrollo , Regiones Promotoras Genéticas/genética , Estructura Terciaria de Proteína , Proteínas Represoras/química , Proteínas Represoras/genética , Somatostatina/genética , Somatostatina/metabolismo , Transactivadores/química , Transactivadores/genética , Factores de Transcripción/metabolismo , Transcripción Genética/genética , Proteínas de Pez CebraRESUMEN
Cell transplantation therapy for diabetes is limited by an inadequate supply of cells exhibiting glucose-responsive insulin secretion. To generate an unlimited supply of human beta-cells, inducibly transformed pancreatic beta-cell lines have been created by expression of dominant oncogenes. The cell lines grow indefinitely but lose differentiated function. Induction of beta-cell differentiation was achieved by stimulating the signaling pathways downstream of the transcription factor PDX-1, cell-cell contact, and the glucagon-like peptide (GLP-1) receptor. Synergistic activation of those pathways resulted in differentiation into functional beta-cells exhibiting glucose-responsive insulin secretion in vitro. Both oncogene-expressing and oncogene-deleted cells were transplanted into nude mice and found to exhibit glucose-responsive insulin secretion in vivo. The ability to grow unlimited quantities of human beta-cells is a major step toward developing a cell transplantation therapy for diabetes.
Asunto(s)
Diferenciación Celular , Insulina/genética , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Factor de Transcripción Activador 1 , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Glucemia/metabolismo , Péptido C/metabolismo , Línea Celular , Trasplante de Células , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas del Ojo , Receptor del Péptido 1 Similar al Glucagón , Glucoquinasa/genética , Glucoquinasa/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Insulina/metabolismo , Secreción de Insulina , Ratones , Ratones Desnudos , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box , Receptores de Glucagón/genética , Receptores de Glucagón/metabolismo , Proteínas Represoras , Somatostatina/genética , Somatostatina/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Trasplante Heterólogo , Regulación hacia ArribaRESUMEN
For more than eighty years, insulin injection has been the only treatment option for all type I and many type II diabetic individuals. Whole pancreas transplantation has been a successful approach for some patients, but is a difficult and complex operation. Recently, it was demonstrated that a glucocorticoid-free immunosuppressive regimen led to remarkably successful islet transplantation. However, both pancreas and islet cell transplantation are limited by the tremendous shortage of cadaveric pancreases that are available for transplantation. Therefore, a major goal of diabetes research is to generate an unlimited source of cells exhibiting glucose-responsive insulin secretion that can be used for transplantation, ideally without the need for systemic immunosuppression. The focus of this review is on how gene therapy can be used in beta cell replacement strategies. Gene transfer to beta cells as well as recent advances in beta cell growth and development will be discussed.
Asunto(s)
Diabetes Mellitus Tipo 1/terapia , Diabetes Mellitus Tipo 2/terapia , Terapia Genética , Trasplante de Islotes Pancreáticos , Animales , Diferenciación Celular , Células Cultivadas , ADN Viral/genética , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Matriz Extracelular/metabolismo , Técnicas de Transferencia de Gen , Vectores Genéticos , Glucosa/metabolismo , Hepatocitos/metabolismo , Humanos , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , ARN Viral/genética , Transducción Genética , Trasplante HeterólogoRESUMEN
Activin A (Act.A), a member of the transforming growth factor beta family of secreted proteins, has been implicated in the regulation of growth and differentiation of various cell types. Betacellulin (BTC), a member of the epidermal growth factor family, converts exocrine AR42J cells to insulin-expressing cells when combined with Act.A. We have used primary cultures of human fetal pancreatic tissue to identify the effects of Act.A and/or BTC on islet development and growth. Exposure to Act.A resulted in a 1.5-fold increase in insulin content (P < 0.005) and a 2-fold increase in the number of cells immunopositive for insulin (P < 0.005). The formation of islet-like cell clusters, containing mainly epithelial cells, during a 5-day culture, was stimulated 1.4-fold by BTC (P < 0.05). BTC alone caused a 2.6-fold increase in DNA synthesis (P < 0.005). These data suggest that Act.A induces endocrine differentiation, whereas BTC has a mitogenic effect on human undifferentiated pancreatic epithelial cells.
Asunto(s)
Sustancias de Crecimiento/fisiología , Inhibinas/fisiología , Péptidos y Proteínas de Señalización Intercelular , Páncreas/crecimiento & desarrollo , Activinas , Betacelulina , Diferenciación Celular , División Celular/fisiología , Células Cultivadas , ADN/biosíntesis , Humanos , Inmunohistoquímica , Insulina/biosíntesis , Insulina/metabolismo , Microscopía Confocal , Páncreas/citología , Páncreas/embriologíaRESUMEN
Cell lines from the fetal and adult pancreas that were developed by retroviral transfer of the SV40T and ras(val12) oncogenes lose insulin expression but retain extremely low levels of somatostatin and glucagon mRNA. In contrast to expanded populations of primary human islet cells, none of them express the homeodomain transcription factor PDX-1. When that factor was expressed in the cell lines by retroviral-mediated gene transfer, one of the cell lines, TRM-6, derived from human fetal islets, exhibited a 10- to 100-fold increase in somatostatin gene expression. This is the first report of induction of the endogenous somatostatin gene by PDX-1. Promotion of cell-cell contact by aggregation of TRM-6/PDX-1 into islet-like clusters produced a further 10- to 100-fold increase in somatostatin mRNA, to a level similar to that of freshly isolated islets, which resulted in production of somatostatin protein. Thus, we demonstrate here that signals induced by cell-cell contact act in synergy with PDX-1 to up-regulate the endogenous somatostatin promoter in an immortalized cell line from human fetal islets. This system provides a powerful model for studying human islet cell development and, particularly, the role of cell-cell contact in the differentiation process.