RESUMEN
OBJECTIVES: Severe congenital diarrhea occurs in approximately half of patients with Aristaless-Related Homeobox (ARX) null mutations. The cause of this diarrhea is unknown. In a mouse model of intestinal Arx deficiency, the prevalence of a subset of enteroendocrine cells is altered, leading to diarrhea. Because polyalanine expansions within the ARX protein are the most common mutations found in ARX-related disorders, we sought to characterize the enteroendocrine population in human tissue of an ARX mutation and in a mouse model of the corresponding polyalanine expansion (Arx). METHODS: Immunohistochemistry and quantitative real-time polymerase chain reaction were the primary modalities used to characterize the enteroendocrine populations. Daily weights were determined for the growth curves, and Oil-Red-O staining on stool and tissue identified neutral fats. RESULTS: An expansion of 7 alanines in the first polyalanine tract of both human ARX and mouse Arx altered enteroendocrine differentiation. In human tissue, cholecystokinin, glucagon-like peptide 1, and somatostatin populations were reduced, whereas the chromogranin A population was unchanged. In the mouse model, cholecystokinin and glucagon-like peptide 1 populations were also lost, although the somatostatin-expressing population was increased. The ARX protein was present in human tissue, whereas the Arx protein was degraded in the mouse intestine. CONCLUSIONS: ARX/Arx is required for the specification of a subset of enteroendocrine cells in both humans and mice. Owing to protein degradation, the Arx mouse recapitulates findings of the intestinal Arx null model, but is not able to further the study of the differential effects of the ARX protein on its transcriptional targets in the intestine.
Asunto(s)
Diarrea/genética , Enfermedades Duodenales/genética , Células Enteroendocrinas/fisiología , Proteínas de Homeodominio/genética , Seudoobstrucción Intestinal/genética , Péptidos/metabolismo , Factores de Transcripción/genética , Adolescente , Animales , Diferenciación Celular/genética , Colecistoquinina/análisis , Cromogranina A/análisis , Diarrea/patología , Modelos Animales de Enfermedad , Enfermedades Duodenales/patología , Duodeno/patología , Células Enteroendocrinas/química , Células Enteroendocrinas/patología , Insuficiencia de Crecimiento/genética , Femenino , Péptido 1 Similar al Glucagón/análisis , Proteínas de Homeodominio/análisis , Humanos , Seudoobstrucción Intestinal/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Mutagénesis Insercional , Somatostatina/análisis , Esteatorrea/genética , Factores de Transcripción/análisisRESUMEN
Enteroendocrine cells secrete over a dozen different hormones responsible for coordinating digestion, absorption, metabolism, and gut motility. Loss of enteroendocrine cells is a known cause of severe congenital diarrhea. Furthermore, enteroendocrine cells regulate glucose metabolism, with the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) playing critical roles in stimulating insulin release by pancreatic ß-cells. Islet1 (Isl1) is a LIM-homeodomain transcription factor expressed specifically in an array of intestinal endocrine cells, including incretin-expressing cells. To examine the impact of intestinal Isl1 on glycemic control, we set out to explore the role of intestinal Isl1 in hormone cell specification and organismal physiology. Mice with intestinal epithelial-specific ablation of Isl1 were obtained by crossing Villin-Cre transgenic animals with mice harboring a Isl1(loxP) allele (Isl1(int) model). Gene ablation of Isl1 in the intestine results in loss of GLP-1, GIP, cholecystokinin (CCK), and somatostatin-expressing cells and an increase in 5-HT (serotonin)-producing cells, while the chromogranin A population was unchanged. This dramatic change in hormonal milieu results in animals with lipid malabsorption and females smaller than their littermate controls. Interestingly, when challenged with oral, not intraperitoneal glucose, the Isl-1 intestinal-deficient animals (Isl1(int)) display impaired glucose tolerance, indicating loss of the incretin effect. Thus the Isl1(int) model confirms that intestinal biology is essential for organism physiology in glycemic control and susceptibility to diabetes.
Asunto(s)
Glucemia/metabolismo , Células Enteroendocrinas/metabolismo , Trastornos del Metabolismo de la Glucosa/metabolismo , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Proteínas con Homeodominio LIM/deficiencia , Factores de Transcripción/deficiencia , Factores de Edad , Animales , Animales Recién Nacidos , Biomarcadores/sangre , Colecistoquinina/metabolismo , Cromogranina A/metabolismo , Diarrea/genética , Diarrea/metabolismo , Grasas de la Dieta/metabolismo , Células Enteroendocrinas/patología , Femenino , Polipéptido Inhibidor Gástrico/metabolismo , Gastrinas/metabolismo , Genotipo , Ghrelina/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , Trastornos del Metabolismo de la Glucosa/sangre , Trastornos del Metabolismo de la Glucosa/genética , Prueba de Tolerancia a la Glucosa , Integrasas/genética , Absorción Intestinal , Mucosa Intestinal/patología , Intestino Delgado/patología , Proteínas con Homeodominio LIM/genética , Síndromes de Malabsorción/genética , Síndromes de Malabsorción/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/genética , Fenotipo , Serotonina/metabolismo , Somatostatina/metabolismo , Factores de Transcripción/genética , Aumento de PesoRESUMEN
Enteroendocrine cells of the gastrointestinal (GI) tract play a central role in metabolism, digestion, satiety and lipid absorption, yet their development remains poorly understood. Here we show that Arx, a homeodomain-containing transcription factor, is required for the normal development of mouse and human enteroendocrine cells. Arx expression is detected in a subset of Neurogenin3 (Ngn3)-positive endocrine progenitors and is also found in a subset of hormone-producing cells. In mice, removal of Arx from the developing endoderm results in a decrease of enteroendocrine cell types including gastrin-, glucagon/GLP-1-, CCK-, secretin-producing cell populations and an increase of somatostatin-expressing cells. This phenotype is also observed in mice with endocrine-progenitor-specific Arx ablation suggesting that Arx is required in the progenitor for enteroendocrine cell development. In addition, depletion of human ARX in developing human intestinal tissue results in a profound deficit in expression of the enteroendocrine cell markers CCK, secretin and glucagon while expression of a pan-intestinal epithelial marker, CDX2, and other non-endocrine markers remained unchanged. Taken together, our findings uncover a novel and conserved role of Arx in mammalian endocrine cell development and provide a potential cause for the chronic diarrhea seen in both humans and mice carrying Arx mutations.
Asunto(s)
Endodermo/embriología , Células Enteroendocrinas/citología , Proteínas de Homeodominio/fisiología , Factores de Transcripción/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular , Linaje de la Célula , Endodermo/citología , Humanos , Ratones , Proteínas del Tejido Nervioso/metabolismoRESUMEN
The contribution of medial calcification to vascular dysfunction in renal failure is unknown. Vascular function was measured ex vivo in control, noncalcified uremic, and calcified uremic aortas from rats with adenine-induced renal failure. Plasma urea was 16 ± 4, 93 ± 14, and 110 ± 25 mg/dl, and aortic calcium content was 27 ± 4, 29 ± 2, and 4,946 ± 1,616 nmol/mg dry wt, respectively, in the three groups. Maximal contraction by phenylephrine (PE) or KCl was reduced 53 and 63% in uremic aortas, and sensitivity to KCl but not PE was increased. Maximal relaxation to acetylcholine was impaired in uremic aortas (30 vs. 65%), and sensitivity to nitroprusside was also reduced, indicating some impairment of endothelium-independent relaxation as well. None of these parameters differed between calcified and noncalcified uremic aortas. However, aortic compliance was reduced in calcified aortas, ranging from 17 to 61% depending on the severity of calcification. We conclude that uremic vascular calcification, even when not severe, significantly reduces arterial compliance. Vascular smooth muscle and endothelial function are altered in renal failure but are not affected by medial calcification, even when severe.
Asunto(s)
Calcinosis/fisiopatología , Circulación Renal/fisiología , Uremia/fisiopatología , Acetilcolina/farmacología , Adenina/farmacología , Animales , Aorta Torácica/citología , Aorta Torácica/efectos de los fármacos , Calcio/metabolismo , Adaptabilidad , Proteínas en la Dieta/farmacología , Endotelio Vascular/fisiología , Masculino , Contracción Muscular/fisiología , Relajación Muscular/fisiología , Músculo Liso/fisiología , Músculo Liso Vascular/fisiopatología , Nitroprusiato/farmacología , Fenilefrina/farmacología , Fósforo Dietético/farmacología , Cloruro de Potasio/farmacología , Ratas , Ratas Sprague-Dawley , Vasoconstrictores/farmacología , Vasodilatadores/farmacologíaRESUMEN
Diastolic heart failure is a major cause of mortality in the elderly population. It is often preceded by diastolic dysfunction, which is characterized by impaired active relaxation and increased stiffness. We tested the hypothesis that senescence-prone (SAMP8) mice would develop diastolic dysfunction compared with senescence-resistant controls (SAMR1). Pulsed-wave Doppler imaging of the ratio of blood flow velocity through the mitral valve during early (E) vs. late (A) diastole was reduced from 1.3 ± 0.03 in SAMR1 mice to 1.2 ± 0.03 in SAMP8 mice (P < 0.05). Tissue Doppler imaging of the early (E') and late (A') diastolic mitral annulus velocities found E' reduced from 25.7 ± 0.9 mm/s in SAMR1 to 21.1 ± 0.8 mm/s in SAMP8 mice and E'/A' similarly reduced from 1.1 ± 0.02 to 0.8 ± 0.03 in SAMR1 vs. SAMP8 mice, respectively (P < 0.05). Invasive hemodynamics revealed an increased slope of the end-diastolic pressure-volume relationship (0.5 ± 0.05 vs. 0.8 ± 0.14; P < 0.05), indicating increased left ventricular chamber stiffness. There were no differences in systolic function or mean arterial pressure; however, diastolic dysfunction was accompanied by increased fibrosis in the hearts of SAMP8 mice. In SAMR1 vs. SAMP8 mice, interstitial collagen area increased from 0.3 ± 0.04 to 0.8 ± 0.09% and perivascular collagen area increased from 1.0 ± 0.11 to 1.6 ± 0.14%. Transforming growth factor-ß and connective tissue growth factor gene expression were increased in the hearts of SAMP8 mice (P < 0.05 for all data). In summary, SAMP8 mice show increased fibrosis and diastolic dysfunction similar to those seen in humans with aging and may represent a suitable model for future mechanistic studies.
Asunto(s)
Envejecimiento Prematuro/complicaciones , Envejecimiento , Insuficiencia Cardíaca/etiología , Miocardio/patología , Disfunción Ventricular Izquierda/etiología , Función Ventricular Izquierda , Factores de Edad , Envejecimiento/genética , Envejecimiento Prematuro/genética , Análisis de Varianza , Animales , Cateterismo Cardíaco , Colágeno/genética , Factor de Crecimiento del Tejido Conjuntivo/genética , Diástole , Modelos Animales de Enfermedad , Ecocardiografía Doppler de Pulso , Elasticidad , Fibrosis , Regulación de la Expresión Génica , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/fisiopatología , Hemodinámica , Ratones , Ratones Transgénicos , Miocardio/metabolismo , ARN Mensajero/metabolismo , Factor de Crecimiento Transformador beta/genética , Disfunción Ventricular Izquierda/genética , Disfunción Ventricular Izquierda/patología , Disfunción Ventricular Izquierda/fisiopatología , Función Ventricular Izquierda/genética , Presión VentricularRESUMEN
Chronic hypoxia contributes to pulmonary hypertension through complex mechanisms that include enhanced NADPH oxidase expression and reactive oxygen species (ROS) generation in the lung. Stimulation of peroxisome proliferator-activated receptor gamma (PPARgamma) reduces the expression and activity of NADPH oxidase. Therefore, we hypothesized that activating PPARgamma with rosiglitazone would attenuate chronic hypoxia-induced pulmonary hypertension, in part, through suppressing NADPH oxidase-derived ROS that stimulate proliferative signaling pathways. Male C57Bl/6 mice were exposed to chronic hypoxia (CH, Fi(O2) 10%) or room air for 3 or 5 weeks. During the last 10 days of exposure, each animal was treated daily by gavage with either the PPARgamma ligand, rosiglitazone (10 mg/kg/d) or with an equal volume of vehicle. CH increased: (1) right ventricular systolic pressure (RVSP), (2) right ventricle weight, (3) thickness of the walls of small pulmonary vessels, (4) superoxide production and Nox4 expression in the lung, and (5) platelet-derived growth factor receptor beta (PDGFRbeta) expression and activity and reduced phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression. Treatment with rosiglitazone prevented the development of pulmonary hypertension at 3 weeks; reversed established pulmonary hypertension at 5 weeks; and attenuated CH-stimulated Nox4 expression and superoxide production, PDGFRbeta activation, and reductions in PTEN expression. Rosiglitazone also attenuated hypoxia-induced increases in Nox4 expression in pulmonary endothelial cells in vitro despite hypoxia-induced reductions in PPARgamma expression. Collectively, these findings indicate that PPARgamma ligands attenuated hypoxia-induced pulmonary vascular remodeling and hypertension by suppressing oxidative and proliferative signals providing novel insights for mechanisms underlying therapeutic effects of PPARgamma activation in pulmonary hypertension.
Asunto(s)
Hipertensión Pulmonar/metabolismo , Hipoxia/metabolismo , PPAR gamma/metabolismo , Transducción de Señal/efectos de los fármacos , Tiazolidinedionas/farmacología , Vasodilatadores/farmacología , Animales , Presión Sanguínea/efectos de los fármacos , Proliferación Celular , Enfermedad Crónica , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/patología , Humanos , Hipertensión Pulmonar/tratamiento farmacológico , Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/patología , Hipoxia/tratamiento farmacológico , Hipoxia/etiología , Hipoxia/patología , Ligandos , Masculino , Ratones , NADPH Oxidasa 4 , NADPH Oxidasas/metabolismo , Tamaño de los Órganos , PPAR gamma/agonistas , Fosfohidrolasa PTEN/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Rosiglitazona , Superóxidos/metabolismoRESUMEN
BACKGROUND: Chronic alcohol consumption perturbs cellular function in a variety of organ systems. Previous studies have suggested that moderate alcohol consumption reduces vascular disease, whereas heavier alcohol consumption may worsen it. The mechanisms for these vascular effects of chronic alcohol ingestion continue to be defined and constitute the focus of this study. METHODS: Male Sprague Dawley rats were fed an isocaloric, Lieber-Decarli liquid diet containing either ethanol (36% calories) or Maltose-Dextrin (substituted for ethanol) for 6 weeks. Telemetric blood pressure measurements were taken before and after ethanol feeding. After the rats were killed, the aortas were analyzed for endothelial nitric oxide (NO) synthase expression and NO production. RESULTS: Chronic ethanol ingestion decreased mean arterial pressure and increased aortic NO production as demonstrated by direct ex vivo measurements using iron diethyldithio-carbamic acid as well as analysis of nitrosyl-hemoglobin (NO-Hb) levels. Consistent with these assays of vascular NO production, endothelium-dependent relaxation responses to acetycholine (Ach) were enhanced in ethanol-fed animals. Aortic endothelial nitric oxide synthase expression was also increased by chronic ethanol ingestion. CONCLUSIONS: These findings demonstrate that a regimen of chronic alcohol ingestion in the rat produced generally salutary effects in the systemic vasculature following a 6-week treatment regimen. These findings extend previous in vitro studies to demonstrate that alcohol has potent effects on vascular endothelial nitric oxide synthase expression, NO production, and vascular function. Consistent with previous reports, these findings confirm that alcohol-induced alterations in the production of reactive nitrogen species play an important role in the pathogenesis of alcohol-mediated tissue effects.
Asunto(s)
Aorta/efectos de los fármacos , Presión Sanguínea/efectos de los fármacos , Depresores del Sistema Nervioso Central/farmacología , Etanol/farmacología , Óxido Nítrico/metabolismo , Animales , Aorta/metabolismo , Células Endoteliales/enzimología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/enzimología , Endotelio Vascular/metabolismo , Masculino , Óxido Nítrico Sintasa de Tipo III/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
The recognition of ß cell dedifferentiation in type 2 diabetes raises the translational relevance of mechanisms that direct and maintain ß cell identity. LIM domain-binding protein 1 (LDB1) nucleates multimeric transcriptional complexes and establishes promoter-enhancer looping, thereby directing fate assignment and maturation of progenitor populations. Many terminally differentiated endocrine cell types, however, remain enriched for LDB1, but its role is unknown. Here, we have demonstrated a requirement for LDB1 in maintaining the terminally differentiated status of pancreatic ß cells. Inducible ablation of LDB1 in mature ß cells impaired insulin secretion and glucose homeostasis. Transcriptomic analysis of LDB1-depleted ß cells revealed the collapse of the terminally differentiated gene program, indicated by a loss of ß cell identity genes and induction of the endocrine progenitor factor neurogenin 3 (NEUROG3). Lineage tracing confirmed that LDB1-depleted, insulin-negative ß cells express NEUROG3 but do not adopt alternate endocrine cell fates. In primary mouse islets, LDB1 and its LIM homeodomain-binding partner islet 1 (ISL1) were coenriched at chromatin sites occupied by pancreatic and duodenal homeobox 1 (PDX1), NK6 homeobox 1 (NKX6.1), forkhead box A2 (FOXA2), and NK2 homeobox 2 (NKX2.2) - factors that co-occupy active enhancers in 3D chromatin domains in human islets. Indeed, LDB1 was enriched at active enhancers in human islets. Thus, LDB1 maintains the terminally differentiated state of ß cells and is a component of active enhancers in both murine and human islets.
Asunto(s)
Diferenciación Celular , Proteínas de Unión al ADN/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas con Dominio LIM/metabolismo , Factores de Transcripción/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Unión al ADN/genética , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Factor Nuclear 3-beta del Hepatocito/genética , Factor Nuclear 3-beta del Hepatocito/metabolismo , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Células Secretoras de Insulina/patología , Proteínas con Dominio LIM/genética , Proteínas con Homeodominio LIM/genética , Proteínas con Homeodominio LIM/metabolismo , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Proteínas de Pez CebraRESUMEN
Islet-1 (Isl-1) is essential for the survival and ensuing differentiation of pancreatic endocrine progenitors. Isl-1 remains expressed in all adult pancreatic endocrine lineages; however, its specific function in the postnatal pancreas is unclear. Here we determine whether Isl-1 plays a distinct role in the postnatal ß-cell by performing physiological and morphometric analyses of a tamoxifen-inducible, ß-cell-specific Isl-1 loss-of-function mouse: Isl-1(L/L); Pdx1-CreER(Tm). Ablating Isl-1 in postnatal ß-cells reduced glucose tolerance without significantly reducing ß-cell mass or increasing ß-cell apoptosis. Rather, islets from Isl-1(L/L); Pdx1-CreER(Tm) mice showed impaired insulin secretion. To identify direct targets of Isl-1, we integrated high-throughput gene expression and Isl-1 chromatin occupancy using islets from Isl-1(L/L); Pdx1-CreER(Tm) mice and ßTC3 insulinoma cells, respectively. Ablating Isl-1 significantly affected the ß-cell transcriptome, including known targets Insulin and MafA as well as novel targets Pdx1 and Slc2a2. Using chromatin immunoprecipitation sequencing and luciferase reporter assays, we found that Isl-1 directly occupies functional regulatory elements of Pdx1 and Slc2a2. Thus Isl-1 is essential for postnatal ß-cell function, directly regulates Pdx1 and Slc2a2, and has a mature ß-cell cistrome distinct from that of pancreatic endocrine progenitors.
Asunto(s)
Resistencia a la Insulina/genética , Células Secretoras de Insulina/metabolismo , Proteínas con Homeodominio LIM/genética , Elementos Reguladores de la Transcripción/genética , Factores de Transcripción/genética , Animales , Apoptosis/genética , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Perfilación de la Expresión Génica , Prueba de Tolerancia a la Glucosa , Transportador de Glucosa de Tipo 2/genética , Transportador de Glucosa de Tipo 2/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Insulina/genética , Insulina/metabolismo , Proteínas con Homeodominio LIM/metabolismo , Factores de Transcripción Maf de Gran Tamaño/genética , Factores de Transcripción Maf de Gran Tamaño/metabolismo , Ratones , Ratones Noqueados , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Pendrin is a Cl-/HCO3- exchanger expressed in the apical regions of renal intercalated cells. Following pendrin gene ablation, blood pressure falls, in part, from reduced renal NaCl absorption. We asked if pendrin is expressed in vascular tissue and if the lower blood pressure observed in pendrin null mice is accompanied by reduced vascular reactivity. Thus, the contractile responses to KCl and phenylephrine (PE) were examined in isometrically mounted thoracic aortas from wild-type and pendrin null mice. Although pendrin expression was not detected in the aorta, pendrin gene ablation changed contractile protein abundance and increased the maximal contractile response to PE when normalized to cross sectional area (CSA). However, the contractile sensitivity to this agent was unchanged. The increase in contractile force/cross sectional area observed in pendrin null mice was due to reduced cross sectional area of the aorta and not from increased contractile force per vessel. The pendrin-dependent increase in maximal contractile response was endothelium- and nitric oxide-independent and did not occur from changes in Ca2+ sensitivity or chronic changes in catecholamine production. However, application of 100 nM angiotensin II increased force/CSA more in aortas from pendrin null than from wild type mice. Moreover, angiotensin type 1 receptor inhibitor (candesartan) treatment in vivo eliminated the pendrin-dependent changes contractile protein abundance and changes in the contractile force/cross sectional area in response to PE. In conclusion, pendrin gene ablation increases aorta contractile force per cross sectional area in response to angiotensin II and PE due to stimulation of angiotensin type 1 receptor-dependent signaling. The angiotensin type 1 receptor-dependent increase in vascular reactivity may mitigate the fall in blood pressure observed with pendrin gene ablation.
Asunto(s)
Angiotensina II/farmacología , Proteínas de Transporte de Anión/genética , Aorta/efectos de los fármacos , Aorta/metabolismo , Transducción de Señal/efectos de los fármacos , Vasoconstricción/efectos de los fármacos , Vasoconstricción/genética , Animales , Proteínas de Transporte de Anión/deficiencia , Aorta/patología , Calcio/metabolismo , Catecolaminas/biosíntesis , Relación Dosis-Respuesta a Droga , Expresión Génica , Riñón/metabolismo , Masculino , Ratones , Ratones Noqueados , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Óxido Nítrico/metabolismo , Fenilefrina/farmacología , Cloruro de Potasio/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor de Angiotensina Tipo 1/metabolismo , Transportadores de Sulfato , Vasoconstrictores/farmacologíaRESUMEN
The specification and differentiation of pancreatic endocrine cell populations (α-, ß-, δ, PP- and ε-cells) is orchestrated by a combination of transcriptional regulators. In the pancreas, Aristaless-related homeobox gene (Arx) is expressed first in the endocrine progenitors and then restricted to glucagon-producing α-cells. While the functional requirement of Arx in early α-cell specification has been investigated, its role in maintaining α-cell identity has yet to be explored. To study this later role of Arx, we have generated mice in which the Arx gene has been ablated specifically in glucagon-producing α-cells. Lineage-tracing studies and immunostaining analysis for endocrine hormones demonstrate that ablation of Arx in neonatal α-cells results in an α-to-ß-like conversion through an intermediate bihormonal state. Furthermore, these Arx-deficient converted cells express ß-cell markers including Pdx1, MafA, and Glut2. Surprisingly, short-term ablation of Arx in adult mice does not result in a similar α-to-ß-like conversion. Taken together, these findings reveal a potential temporal requirement for Arx in maintaining α-cell identity.
Asunto(s)
Eliminación de Gen , Células Secretoras de Glucagón/metabolismo , Proteínas de Homeodominio/genética , Factores de Transcripción/genética , Animales , Animales Recién Nacidos , Biomarcadores/metabolismo , Linaje de la Célula/genética , Femenino , Expresión Génica , Glucagón/genética , Glucagón/metabolismo , Insulina/genética , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Masculino , Ratones , Ratones TransgénicosRESUMEN
Pulmonary arterial hypertension (PAH) is a progressive disease characterized by increased pulmonary arterial resistance and vessel remodeling. Patients living with human immunodeficiency virus-1 (HIV-1) have an increased susceptibility to develop severe pulmonary hypertension (PH) irrespective of their CD4+ lymphocyte counts. While the underlying cause of HIV-PAH remains unknown, the interaction of HIV-1 proteins with the vascular endothelium may play a critical role in HIV-PAH development. Hypoxia promotes PH in experimental models and in humans, but the impact of HIV-1 proteins on hypoxia-induced pulmonary vascular dysfunction and PAH has not been examined. Therefore, we hypothesize that the presence of HIV-1 proteins and hypoxia synergistically augment the development of pulmonary vascular dysfunction and PH. We examined the effect of HIV-1 proteins on pulmonary vascular resistance by measuring pressure-volume relationships in isolated lungs from wild-type (WT) and HIV-1 Transgenic (Tg) rats. WT and HIV-1 Tg rats were exposed to 10% O2 for four weeks to induce experimental pulmonary hypertension to assess whether HIV-1 protein expression would impact the development of hypoxia-induced PH. Our results demonstrate that HIV-1 protein expression significantly increased pulmonary vascular resistance (PVR). HIV-1 Tg mice demonstrated exaggerated pulmonary vascular responses to hypoxia as evidenced by greater increases in right ventricular systolic pressures, right ventricular hypertrophy and vessel muscularization when compared to wild-type controls. This enhanced PH was associated with enhanced expression of HIF-1α and PCNA. In addition, in vitro studies reveal that medium from HIV-infected monocyte derived macrophages (MDM) potentiates hypoxia-induced pulmonary artery endothelial proliferation. These results indicate that the presence of HIV-1 proteins likely impact pulmonary vascular resistance and exacerbate hypoxia-induced PH.
RESUMEN
A decrease in the expression of Islet-1 (Isl-1), an islet transcription factor, has been reported in several physiological settings of reduced ß-cell function. Here, we investigate whether an increased level of Isl-1 in islet cells can enhance ß-cell function and/or mass. We demonstrate that transgenic mice with Isl-1 overexpression display improved glucose tolerance and enhanced insulin secretion without significant changes in ß cell mass. From our microarray study, we identify approximately 135 differentially expressed genes in the islets of Isl-1 overexpressing mice that have been implicated to function in numerous biological processes including protein trafficking, metabolism and differentiation. Using real-time PCR we have confirmed upregulation of Caps2, Sec14l4, Slc2a10, P2rx7, Afamin, and Neurogenin 3 that may in part mediate the observed improved insulin secretion in Isl-1 overexpressing mice. These findings show for the first time that Isl-1 is a key factor in regulating adult ß cell function in vivo, and suggest that Isl-1 elevation could be beneficial to improve glucose homeostasis.
Asunto(s)
Células Secretoras de Insulina/fisiología , Proteínas con Homeodominio LIM/metabolismo , Factores de Transcripción/metabolismo , Animales , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Glucosa/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN/química , ARN/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Nucleoside reverse transcriptase inhibitors (NRTIs), such as zidovudine (AZT) and stavudine (d4T), cause toxicities to numerous tissues, including the liver and vasculature. While much is known about hepatic NRTI toxicity, the mechanism of toxicity in endothelial cells is incompletely understood. Human aortic endothelial and HepG2 liver cells were exposed to 1 muM AZT or d4T for up to 5 weeks. Markers of oxidative stress, mitochondrial function, NRTI phosphorylation, mitochondrial DNA (mtDNA) levels, and cytotoxicity were monitored over time. In endothelial cells, AZT significantly oxidized glutathione redox potential, increased total cellular and mitochondrial-specific superoxide, decreased mitochondrial membrane potential, increased lactate release, and caused cell death from weeks 3 through 5. Toxicity occurred in the absence of di- and tri-phosphorylated AZT and mtDNA depletion. These data show that oxidative stress and mitochondrial dysfunction in endothelial cells occur with a physiologically relevant concentration of AZT, and require long-term exposure to develop. In contrast, d4T did not induce endothelial oxidative stress, mitochondrial dysfunction, or cytotoxicity despite the presence of d4T-triphosphate. Both drugs depleted mtDNA in HepG2 cells without causing cell death. Endothelial cells are more susceptible to AZT-induced toxicity than HepG2 cells, and AZT caused greater endothelial dysfunction than d4T because of its pro-oxidative effects.