RESUMEN

Type 1 diabetes (T1DM) results from destruction of most insulin-secreting pancreatic ß-cells. The persistence of ß-cells decades after the onset of the disease indicates that the resistance of individual cells to the autoimmune insult is heterogeneous and might depend on the metabolic status of a cell at a given moment. The aim of this study is to investigate whether activation of nicotinic acetylcholine receptors (nACh-Rs) could increase ß-cell resistance against the adverse environment prevailing at the onset of T1DM. Here, we show that nACh-R activation by nicotine and choline, 2 agonists of the receptor, decreases murine and human ß-cell apoptosis induced by proinflammatory cytokines known to be present in the islet environment at the onset of T1DM. The protective mechanism activated by nicotine and choline involves attenuation of mitochondrial outer membrane permeabilization via modulation of endoplasmic reticulum stress, of the activity of B-cell lymphoma 2 family proteins and cytoplasmic calcium levels. Local inflammation and endoplasmic reticulum stress being key determinants of ß-cell death in T1DM, we conclude that pharmacological activation of nACh-R could represent a valuable therapeutic option in the modulation of ß-cell death in T1DM.

Asunto(s)

Apoptosis/efectos de los fármacos , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Islotes Pancreáticos/efectos de los fármacos , Agonistas Nicotínicos/uso terapéutico , Animales , Calcio/metabolismo , Caspasa 3/metabolismo , Colina , Técnicas de Cultivo , Evaluación Preclínica de Medicamentos , Estrés del Retículo Endoplásmico , Femenino , Regulación de la Expresión Génica , Genes bcl-2 , Humanos , Ratones , Ratones Endogámicos C57BL , Membranas Mitocondriales/metabolismo , Nicotina , Agonistas Nicotínicos/farmacología , Óxido Nítrico/metabolismo

RESUMEN

The Forkhead box A transcription factors are major regulators of glucose homeostasis. They show both distinct and redundant roles during pancreas development and in adult mouse ß-cells. In vivo ablation studies have revealed critical implications of Foxa1 on glucagon biosynthesis and requirement of Foxa2 in α-cell terminal differentiation. In order to examine the respective role of these factors in mature α-cells, we used small interfering RNA (siRNA) directed against Foxa1 and Foxa2 in rat primary pancreatic α-cells and rodent α-cell lines leading to marked decreases in Foxa1 and Foxa2 mRNA levels and proteins. Both Foxa1 and Foxa2 control glucagon gene expression specifically through the G2 element. Although we found that Foxa2 controls the expression of the glucagon, MafB, Pou3f4, Pcsk2, Nkx2.2, Kir6.2, and Sur1 genes, Foxa1 only regulates glucagon gene expression. Interestingly, the Isl1 and Gipr genes were not controlled by either Foxa1 or Foxa2 alone but by their combination. Foxa1 and Foxa2 directly activate and bind the promoter region the Nkx2.2, Kir6.2 and Sur1, Gipr, Isl1, and Pou3f4 genes. We also demonstrated that glucagon secretion is affected by the combined effects of Foxa1 and Foxa2 but not by either one alone. Our results indicate that Foxa1 and Foxa2 control glucagon biosynthesis and secretion as well as α-cell differentiation with both common and unique target genes.

Asunto(s)

Diferenciación Celular/genética , Células Secretoras de Glucagón/fisiología , Glucagón/biosíntesis , Glucagón/metabolismo , Factor Nuclear 3-alfa del Hepatocito/fisiología , Factor Nuclear 3-beta del Hepatocito/fisiología , Animales , Sitios de Unión/genética , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Cricetinae , Factor Nuclear 3-alfa del Hepatocito/antagonistas & inhibidores , Factor Nuclear 3-beta del Hepatocito/antagonistas & inhibidores , Proteína Homeobox Nkx-2.2 , Masculino , Regiones Promotoras Genéticas , ARN Interferente Pequeño/farmacología , Ratas

RESUMEN

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated cation channels well characterized in neuronal signal transmission. Moreover, recent studies have revealed nAChR expression in nonneuronal cell types throughout the body, including tissues involved in metabolism. In the present study, we screen gene expression of nAChR subunits in pancreatic islets and adipose tissues. Mice pancreatic islets present predominant expression of α7 and ß2 nAChR subunits but at a lower level than in central structures. Characterization of glucose and energy homeostasis in α7ß2nAChR(-/-) mice revealed no major defect in insulin secretion and sensitivity but decreased glycemia apparently unrelated to gluconeogenesis or glycogenolysis. α7ß2nAChR(-/-) mice presented an increase in lean and bone body mass and a decrease in fat storage with normal body weight. These observations were associated with elevated spontaneous physical activity in α7ß2nAChR(-/-) mice, mainly due to elevation in fine vertical (rearing) activity while their horizontal (ambulatory) activity remained unchanged. In contrast to α7nAChR(-/-) mice presenting glucose intolerance and insulin resistance associated to excessive inflammation of adipose tissue, the present metabolic phenotyping of α7ß2nAChR(-/-) mice revealed a metabolic improvement possibly linked to the increase in spontaneous physical activity related to central ß2nAChR deficiency.

Asunto(s)

Tejido Adiposo/metabolismo , Glucosa/metabolismo , Islotes Pancreáticos/metabolismo , Receptores Nicotínicos/metabolismo , Animales , Índice Glucémico , Humanos , Insulina/metabolismo , Secreción de Insulina , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora , Receptores Nicotínicos/genética

RESUMEN

Poor fetal growth, also known as intrauterine growth restriction (IUGR), is a worldwide health concern. IUGR is commonly associated with both an increased risk in perinatal mortality and a higher prevalence of developing chronic metabolic diseases later in life. Obesity, type 2 diabetes or metabolic syndrome could result from noxious "metabolic programming." In order to better understand early alterations involved in metabolic programming, we modeled IUGR rat pups through either prenatal exposure to synthetic glucocorticoid (dams infused with dexamethasone 100 µg/kg/day, DEX) or prenatal undernutrition (dams feeding restricted to 30% of ad libitum intake, UN). Physiological (glucose and insulin tolerance), morphometric (automated tissue image analysis) and transcriptomic (quantitative PCR) approaches were combined during early life of these IUGR pups with a special focus on their endocrine pancreas and adipose tissue development. In the absence of catch-up growth before weaning, DEX and UN IUGR pups both presented basal hyperglycaemia, decreased glucose tolerance, and pancreatic islet atrophy. Other early metabolic defects were model-specific: DEX pups presented decreased insulin sensitivity whereas UN pups exhibited lowered glucose-induced insulin secretion and more marked alterations in gene expression of pancreatic islet and adipose tissue development regulators. In conclusion, these results show that before any catch-up growth, IUGR rats present early physiologic, morphologic and transcriptomic defects, which can be considered as initial mechanistic basis of metabolic programming.

Asunto(s)

Dexametasona/efectos adversos , Retardo del Crecimiento Fetal/metabolismo , Desnutrición/metabolismo , Efectos Tardíos de la Exposición Prenatal/metabolismo , Tejido Adiposo/crecimiento & desarrollo , Tejido Adiposo/metabolismo , Análisis de Varianza , Animales , Glucemia/metabolismo , Western Blotting , Pesos y Medidas Corporales , Péptido C/sangre , Corticosterona/sangre , Cartilla de ADN/genética , Femenino , Perfilación de la Expresión Génica , Insulina/sangre , Resistencia a la Insulina/fisiología , Islotes Pancreáticos/crecimiento & desarrollo , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Leptina/sangre , Embarazo , Radioinmunoensayo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa

RESUMEN

The Pax6 transcription factor is crucial for pancreatic α-cells. Indeed, Pax6-deficient mouse models are characterized by markedly altered α-cell differentiation. Our objective was to investigate the role of Pax6 in glucagon secretion process. We used a Pax6-deficient model in rat primary enriched-α cells with specific small interfering RNA leading to a 70% knockdown of Pax6 expression. We first showed that Pax6 knockdown decreases glucagon biosynthesis as well as glucagon release. Through physiological assays, we demonstrated that the decrease of Pax6 affects specifically acute glucagon secretion in primary α-cell in response to glucose, palmitate, and glucose-dependent insulinotropic peptide (GIP) but not the response to arginine and epinephrine. We identified in Pax6 knockdown model that genes involved in glucagon secretion such as the glucokinase (GCK), G protein-coupled receptor (GPR40), and GIP receptor (GIPR) as well as the corresponding proteins were significantly decreased whereas the insulin receptor (IR) Kir6.2/Sur1, and glucose transporter 1 genes were not affected. We demonstrated that Pax6 directly binds and activates specific elements on the promoter region of the GPR40, GCK, and GIPR genes. Finally, through site-directed mutagenesis experiments, we showed that disruption of Pax6 binding on the GCK, GPR40, and GIPR gene promoters led to specific decreases of their activities in the αTC1.9 glucagon-producing cell line. Hence our results indicate that Pax6 acts on the regulation of glucagon secretion at least through the transcriptional control of GCK, GPR40, and GIPR. We propose that Pax6 is not only critical for glucagon biosynthesis but also for glucagon secretion particularly in response to nutrients.

Asunto(s)

Proteínas del Ojo/metabolismo , Glucagón/metabolismo , Proteínas de Homeodominio/metabolismo , Factores de Transcripción Paired Box/metabolismo , Proteínas Represoras/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Células Cultivadas , Proteínas del Ojo/genética , Glucoquinasa/genética , Glucoquinasa/metabolismo , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 1/metabolismo , Proteínas de Homeodominio/genética , Inmunoprecipitación , Mutagénesis Sitio-Dirigida , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/genética , Canales de Potasio de Rectificación Interna/genética , Canales de Potasio de Rectificación Interna/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica , Ratas , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Receptores de Droga/genética , Receptores de Droga/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de la Hormona Gastrointestinal/genética , Receptores de la Hormona Gastrointestinal/metabolismo , Proteínas Represoras/genética , Receptores de Sulfonilureas

RESUMEN

Abnormal adipokine production, along with defective uptake and metabolism of glucose within adipocytes, contributes to insulin resistance and altered glucose homeostasis. Recent research has highlighted one of the mechanisms that accounts for impaired production of adiponectin (ADIPOQ) and adipocyte glucose uptake in obesity. In adipocytes of human obese subjects and mice fed with a high fat diet, the level of the inducible cAMP early repressor (ICER) is diminished. Reduction of ICER elevates the cAMP response element binding protein (CREB) activity, which in turn increases the repressor activating transcription factor 3. In fine, the cascade triggers reduction in the ADIPOQ and GLUT4 levels, which ultimately hampers insulin-mediated glucose uptake. The c-Jun N-terminal kinase (JNK) interacting-protein 1, also called islet brain 1 (IB1), is a target of CREB/ICER that promotes JNK-mediated insulin resistance in adipocytes. A rise in IB1 and c-Jun levels accompanies the drop of ICER in white adipose tissues of obese mice when compared with mice fed with a chow diet. Other than the expression of ADIPOQ and glucose transport, decline in ICER expression might impact insulin signaling. Impairment of ICER is a critical issue that will need major consideration in future therapeutic purposes.

RESUMEN

The paired box homeodomain Pax6 is crucial for endocrine cell development and function and plays an essential role in glucose homeostasis. Indeed, mutations of Pax6 are associated with diabetic phenotype. Importantly, homozygous mutant mice for Pax6 are characterized by markedly decreased ß and δ cells and absent α cells. To better understand the critical role that Pax6 exerts in glucagon-producing cells, we developed a model of primary rat α cells. To study the transcriptional network of Pax6 in adult and differentiated α cells, we generated Pax6-deficient primary rat α cells and glucagon-producing cells, using either specific siRNA or cells expressing constitutively a dominant-negative form of Pax6. In primary rat α cells, we confirm that Pax6 controls the transcription of the Proglucagon and processing enzyme PC2 genes and identify three new target genes coding for MafB, cMaf, and NeuroD1/Beta2, which are all critical for Glucagon gene transcription and α cell differentiation. Furthermore, we demonstrate that Pax6 directly binds and activates the promoter region of the three genes through specific binding sites and that constitutive expression of a dominant-negative form of Pax6 in glucagon-producing cells (InR1G9) inhibits the activities of the promoters. Finally our results suggest that the critical role of Pax6 action on α cell differentiation is independent of those of Arx and Foxa2, two transcription factors that are necessary for α cell development. We conclude that Pax6 is critical for α cell function and differentiation through the transcriptional control of key genes involved in glucagon gene transcription, proglucagon processing, and α cell differentiation.

Asunto(s)

Diferenciación Celular/fisiología , Proteínas del Ojo/metabolismo , Regulación de la Expresión Génica/fisiología , Células Secretoras de Glucagón/metabolismo , Proteínas de Homeodominio/metabolismo , Modelos Biológicos , Factores de Transcripción Paired Box/metabolismo , Proteínas Represoras/metabolismo , Elementos de Respuesta/fisiología , Animales , Línea Celular , Proteínas del Ojo/genética , Células Secretoras de Glucagón/citología , Proteínas de Homeodominio/genética , Humanos , Ratones , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/genética , Proglucagón/biosíntesis , Proglucagón/genética , Ratas , Proteínas Represoras/genética

RESUMEN

Mutations in PCSK1 cause monogenic obesity. To assess the contribution of PCSK1 to polygenic obesity risk, we genotyped tag SNPs in a total of 13,659 individuals of European ancestry from eight independent case-control or family-based cohorts. The nonsynonymous variants rs6232, encoding N221D, and rs6234-rs6235, encoding the Q665E-S690T pair, were consistently associated with obesity in adults and children (P = 7.27 x 10(-8) and P = 2.31 x 10(-12), respectively). Functional analysis showed a significant impairment of the N221D-mutant PC1/3 protein catalytic activity.

Asunto(s)

Predisposición Genética a la Enfermedad , Obesidad/genética , Polimorfismo de Nucleótido Simple , Proproteína Convertasa 1/genética , Adulto , Estudios de Casos y Controles , Niño , Humanos , Obesidad/metabolismo , Proproteína Convertasa 1/metabolismo , Población Blanca

RESUMEN

The cocaine and amphetamine regulated transcript (CART), an anorexigenic peptide responding to leptin, is expressed in various areas of the hypothalamus. The role of CART in humans and its potential contribution to abnormalities in feeding control are mostly unknown. Since CART plays an important role in the hypothalamic regulation of energy balance by reducing food intake and increasing lipid substrate utilization, it might affect cholesterol metabolism as Neuropeptide Y or pro-opiomelanocortin do. In the present work, we studied the potential effects of three SNPs of the CART promoter in a WHO-MONICA general population from North of France (n=840), untreated for hypercholesterolemia, hypertension, or diabetes mellitus since any treatment is likely to interfere with lipoprotein/lipid variables. Our results show associations between these SNPs and plasma LDL-cholesterol level and the LDL/HDL ratio, a marker of atherogenicity. A haplotypic study suggests that these effects are mainly attributable to the functional SNP -3608C>T. Subjects bearing the -3608 C allele present a plasma lipid profile protective against atherogenesis: decrease of plasma LDL-cholesterol level (p=0.001) and of the LDL/HDL ratio (p=0.0003). This result offers new evidences for a potential implication of the CART gene in lipid metabolism and in atherogenesis.

Asunto(s)

Variación Genética , Lípidos/sangre , Proteínas del Tejido Nervioso/genética , Regiones Promotoras Genéticas , Adulto , Anciano , Colesterol/sangre , Femenino , Genética de Población , Haplotipos/genética , Humanos , Resistencia a la Insulina , Leptina/sangre , Lípidos/genética , Lipoproteínas HDL/sangre , Lipoproteínas HDL/genética , Lipoproteínas LDL/sangre , Lipoproteínas LDL/genética , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple

Asunto(s)

Remodelación Ósea/genética , Proteínas del Tejido Nervioso/genética , Obesidad/genética , Anciano , Anciano de 80 o más Años , Femenino , Predisposición Genética a la Enfermedad , Humanos , Persona de Mediana Edad , Polimorfismo Genético , Posmenopausia/genética

RESUMEN

BACKGROUND: Cocaine and amphetamine regulated transcript (CART) is an anorectic neuropeptide located principally in hypothalamus. CART has been shown to be involved in control of feeding behavior, but a direct relationship with obesity has not been established. The aim of this study was to evaluate the effect of polymorphisms within the CART gene with regards to a possible association with obesity in a Caucasian population. RESULTS: Screening of the entire gene as well as a 3.7 kb region of 5' upstream sequence revealed 31 SNPs and 3 rare variants; 14 of which were subsequently genotyped in 292 French morbidly obese subjects and 368 controls. Haplotype analysis suggested an association with obesity which was found to be mainly due to SNP-3608T>C (rs7379701) (p = 0.009). Genotyping additional cases and controls also of European Caucasian origin supported further this possible association between the CART SNP -3608T>C T allele and obesity (global p-value = 0.0005). Functional studies also suggested that the SNP -3608T>C could modulate nuclear protein binding. CONCLUSION: CART SNP -3608T>C may possibly contribute to the genetic risk for obesity in the Caucasian population. However confirmation of the importance of the role of the CART gene in energy homeostasis and obesity will require investigation and replication in further populations.

Asunto(s)

Proteínas del Tejido Nervioso/genética , Obesidad/genética , Polimorfismo Genético , Población Blanca/genética , Secuencia de Bases , Estudios de Casos y Controles , Genotipo , Humanos , Polimorfismo de Nucleótido Simple , Factores de Riesgo