RESUMEN
Insulin resistance and ß-cell failure are the major defects in type 2 diabetes mellitus. However, the molecular mechanisms linking these two defects remain unknown. Elevated levels of apolipoprotein CIII (apoCIII) are associated not only with insulin resistance but also with cardiovascular disorders and inflammation. We now demonstrate that local apoCIII production is connected to pancreatic islet insulin resistance and ß-cell failure. An increase in islet apoCIII causes promotion of a local inflammatory milieu, increased mitochondrial metabolism, deranged regulation of ß-cell cytoplasmic free Ca(2+) concentration ([Ca(2+)]i) and apoptosis. Decreasing apoCIII in vivo results in improved glucose tolerance, and pancreatic apoCIII knockout islets transplanted into diabetic mice, with high systemic levels of the apolipoprotein, demonstrate a normal [Ca(2+)]i response pattern and no hallmarks of inflammation. Hence, under conditions of islet insulin resistance, locally produced apoCIII is an important diabetogenic factor involved in impairment of ß-cell function and may thus constitute a novel target for the treatment of type 2 diabetes mellitus.
Asunto(s)
Apolipoproteína C-III/metabolismo , Diabetes Mellitus Tipo 2/fisiopatología , Resistencia a la Insulina/fisiología , Células Secretoras de Insulina/patología , Análisis de Varianza , Animales , Apolipoproteína C-III/genética , Western Blotting , Calcio/metabolismo , Línea Celular Tumoral , Inmunohistoquímica , Ratones , Ratones Noqueados , Microscopía Confocal , Mitocondrias/metabolismo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
AIMS/HYPOTHESIS: In vivo imaging of the developing pancreas is challenging due to the inaccessibility of the tissue. To circumvent this, on embryonic day 10.5 (E10.5) we transplanted a mouse developing pancreatic bud into the anterior chamber of the eye (ACE) to determine whether the eye is a useful transplant site to support pancreas development. METHODS: We transplanted an E10.5 dorsal pancreatic bud into the ACE of a syngeneic recipient mouse. Using a mouse insulin promoter-green fluorescent protein (MIP-GFP) mouse as the tissue donor, we non-invasively imaged the pancreatic bud as it develops at single beta cell resolution across time. RESULTS: The transplanted pancreatic bud rapidly engrafts and vascularises when transplanted into the ACE. The pancreatic progenitor cells differentiate into exocrine and endocrine cells, including cells expressing insulin, glucagon and somatostatin. The morphology of the transplanted pancreatic bud resembles that of the native developing pancreas. Beta cells within the transplanted pancreatic bud respond to glucose in a manner similar to that of native fetal beta cells and superior to that of in vitro developed beta cells. Unlike in vitro grown pancreatic explants, pancreatic tissue developing in the ACE is vascularised, providing the developing pancreatic tissue with a milieu resembling the native situation. CONCLUSIONS/INTERPRETATION: Altogether, we show that the ACE is able to support growth, differentiation and function of a developing pancreatic bud across time in vivo.
Asunto(s)
Cámara Anterior/citología , Islotes Pancreáticos/embriología , Páncreas/embriología , Animales , Cámara Anterior/metabolismo , Diferenciación Celular/fisiología , Femenino , Insulina/metabolismo , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Masculino , Ratones , EmbarazoRESUMEN
Inflammation and impaired mitochondrial oxidative phosphorylation are considered key players in the development of several metabolic disorders, including diabetes. We have previously shown inflammation and mitochondrial dysfunction in the hypothalamus of an animal model for anorexia, the anx/anx mouse. Moreover, increased incidence of eating disorders, e.g., anorexia nervosa, has been observed in diabetic individuals. In the present investigation we evaluated whether impaired mitochondrial phosphorylation and inflammation also occur in endocrine pancreas of anorectic mice, and if glucose homeostasis is disturbed. We show that anx/anx mice exhibit marked glucose intolerance associated with reduced insulin release following an intraperitoneal injection of glucose. In contrast, insulin release from isolated anx/anx islets is increased after stimulation with glucose or KCl. In isolated anx/anx islets there is a strong downregulation of the mitochondrial complex I (CI) assembly factor, NADH dehydrogenase (ubiquinone) 1α subcomplex, assembly factor 1 (Ndufaf1), and a reduced CI activity. In addition, we show elevated concentrations of free fatty acids (FFAs) in anx/anx serum and increased macrophage infiltration (indicative of inflammation) in anx/anx islets. However, isolated islets from anx/anx mice cultured in the absence of FFAs do not exhibit increased inflammation. We conclude that the phenotype of the endocrine pancreas of the anx/anx mouse is characterized by increased levels of circulating FFAs, as well as inflammation, which can inhibit insulin secretion in vivo. The anx/anx mouse may represent a useful tool for studying molecular mechanisms underlying the association between diabetes and eating disorders.
Asunto(s)
Anorexia/fisiopatología , Intolerancia a la Glucosa/fisiopatología , Células Secretoras de Insulina/fisiología , Animales , Anorexia/complicaciones , Anorexia/metabolismo , Anorexia/patología , Glucemia/metabolismo , Recuento de Células , Células Cultivadas , Intolerancia a la Glucosa/complicaciones , Intolerancia a la Glucosa/metabolismo , Intolerancia a la Glucosa/patología , Prueba de Tolerancia a la Glucosa , Insulina/sangre , Células Secretoras de Insulina/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Tamaño de los Órganos , Páncreas/patologíaRESUMEN
Liver receptor homolog 1 (LRH-1) and pancreatic-duodenal homeobox 1 (PDX-1) are coexpressed in the pancreas during mouse embryonic development. Analysis of the regulatory region of the human LRH-1 gene demonstrated the presence of three functional binding sites for PDX-1. Electrophoretic mobility shift assays and chromatin immunoprecipitation analysis showed that PDX-1 bound to the LRH-1 promoter, both in cultured cells in vitro and during pancreatic development in vivo. Retroviral expression of PDX-1 in pancreatic cells induced the transcription of LRH-1, whereas reduced PDX-1 levels by RNA interference attenuated its expression. Consistent with direct regulation of LRH-1 expression by PDX-1, PDX-1(-/-) mice expressed smaller amounts of LRH-1 mRNA in the embryonic pancreas. Taken together, our data indicate that PDX-1 controls LRH-1 expression and identify LRH-1 as a novel downstream target in the PDX-1 regulatory cascade governing pancreatic development, differentiation, and function.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio , Hígado/metabolismo , Páncreas/embriología , Receptores Citoplasmáticos y Nucleares/genética , Transactivadores/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Secuencia Conservada , Humanos , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Páncreas/metabolismo , Regiones Promotoras Genéticas , Receptores Citoplasmáticos y Nucleares/metabolismo , Homología de Secuencia de Aminoácido , Transactivadores/genética , Células Tumorales CultivadasRESUMEN
Stem cell research and the prospect of stem cell based therapies depend critically on the identification of specific markers that can be used for the identification and selection of stem and progenitor cells. Nestin is expressed in neuronal progenitor cells and has also been suggested to mark multipotent pancreatic stem cells. We show here that, throughout pancreatic development, markers of pancreatic progenitor cells and differentiated pancreatic cells are expressed in E-cadherin-positive epithelial cells that do not express nestin. The data presented demonstrate that nestin is expressed in mesenchymal and not epithelial cells of the developing mouse pancreas.
Asunto(s)
Células Epiteliales/metabolismo , Proteínas de Filamentos Intermediarios/biosíntesis , Mesodermo/metabolismo , Proteínas del Tejido Nervioso , Páncreas/embriología , Animales , Cadherinas/biosíntesis , Inmunohistoquímica , Ratones , Microscopía Confocal , Nestina , Factores de TiempoRESUMEN
The identification of secreted factors that can selectively stimulate the generation of insulin producing beta-cells from stem and/or progenitor cells represent a significant step in the development of stem cell-based beta-cell replacement therapy. By elucidating the molecular mechanisms that regulate the generation of beta-cells during normal pancreatic development such putative factors may be identified. In the mouse, beta-cells increase markedly in numbers from embryonic day (e) 14.5 and onwards, but the extra-cellular signal(s) that promotes the selective generation of beta-cells at these stages remains to be identified. Here we show that the retinoic acid (RA) synthesizing enzyme Raldh1 is expressed in developing mouse and human pancreas at stages when beta-cells are generated. We also provide evidence that RA induces the generation of Ngn3(+) endocrine progenitor cells and stimulates their further differentiation into beta-cells by activating a program of cell differentiation that recapitulates the normal temporal program of beta-cell differentiation.
Asunto(s)
Sistema Endocrino/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células Secretoras de Insulina/citología , Páncreas/metabolismo , Células Madre/citología , Tretinoina/metabolismo , Aldehído Deshidrogenasa/metabolismo , Familia de Aldehído Deshidrogenasa 1 , Animales , Diferenciación Celular , Humanos , Hibridación in Situ , Ratones , Ratones Transgénicos , Páncreas/embriología , Retinal-Deshidrogenasa , Transducción de Señal , Factores de TiempoRESUMEN
The transcription factor IPF1/PDX1 plays a crucial role in both pancreas development and maintenance of beta-cell function. Targeted disruption of this transcription factor in beta-cells leads to diabetes, whereas reduced expression levels affect insulin expression and secretion. Therefore, it is essential to determine molecular mechanisms underlying the regulation of this key transcription factor on mRNA levels and, most importantly, on protein levels. Here we show that a minor portion of IPF1/PDX1 is phosphorylated on serine 61 and/or serine 66 in pancreatic beta-cells. This phosphorylated form of IPF1/PDX1 preferentially accumulates following proteasome inhibition, an effect that is prevented by inhibition of glycogen synthase kinase 3 (GSK3) activity. Oxidative stress, which is associated with the diabetic state, (i) increases IPF1/PDX1 Ser61 and/or Ser66 phosphorylation and (ii) increases the degradation rate and decreases the half-life of IPF-1/PDX-1 protein. In addition, we provide evidence that GSK3 activity participates in oxidative stress-induced effects on beta-cells. Thus, this current study uncovers a new mechanism that might contribute to diminished levels of IPF1/PDX1 protein and beta-cell dysfunction during the progression of diabetes.