RESUMEN
Proglucagon is cleaved to glucagon by prohormone convertase 2 (PC2) in pancreatic α-cells, but is cleaved to glucagon-like peptide-1 (GLP-1) by PC1 in intestinal L-cells. The aim of this study was to identify mechanisms which switch processing of proglucagon to generate GLP-1 in the pancreas, given that GLP-1 can increase insulin secretion and ß-cell mass. The α-cell line, αTC1-6, expressed PC1 at low levels and GLP-1 was detected in cells and in culture media. GLP-1 was also found in isolated human islets and in rat islets cultured for 7 days. High glucose concentrations increased Pc1 gene expression and PC1 protein in rat islets. High glucose (25â mM) also increased GLP-1 but decreased glucagon secretion from αTC1-6 cells suggesting a switch in processing to favour GLP-1. Three G protein-coupled receptors, GPR120, TGR5 and GPR119, implicated in the release of GLP-1 from L-cells are expressed in αTC1-6 cells. Incubation of these cells with an agonist of TGR5 increased PC1 promoter activity and GLP-1 secretion suggesting that this is a mechanism for switching processing to GLP-1 in the pancreas. Treatment of isolated rat islets with streptozotocin caused ß-cell toxicity as evidenced by decreased glucose-stimulated insulin secretion. This increased GLP-1 but not glucagon in the islets. In summary, proglucagon can be processed to GLP-1 in pancreatic cells. This process is upregulated by elevated glucose, activation of TGR5 and ß-cell destruction. Understanding this phenomenon may lead to advances in therapies to protect ß-cell mass, and thereby slow progression from insulin resistance to type 2 diabetes.
Asunto(s)
Péptido 1 Similar al Glucagón/metabolismo , Células Secretoras de Glucagón/metabolismo , Células Secretoras de Insulina/metabolismo , Comunicación Paracrina/fisiología , Proglucagón/metabolismo , Animales , Línea Celular , Células Cultivadas , Glucagón/metabolismo , Células Secretoras de Glucagón/citología , Células Secretoras de Glucagón/efectos de los fármacos , Glucosa/farmacología , Humanos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Islotes Pancreáticos/citología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Masculino , Ratones , Modelos Animales , Proproteína Convertasa 1/metabolismo , Ratas , Ratas WistarRESUMEN
INTRODUCTION: Leptin deficiency caused by mutations within the leptin gene (LEP) results in severe early onset obesity, hypogonadism, pubertal delay and immune system abnormalities. Constitutional delay in growth and puberty (CDGP) is a common condition seen in paediatric clinics, in which children present with delayed growth and puberty but usually also have a slim body habitus. We hypothesized that LEP variants may play a role in the phenotype seen in CDGP. AIM: To screen a group of children with CDGP for pathogenic sequence variants in LEP. PATIENTS AND METHODS: Denaturing HPLC was used to screen for LEP sequence variants in DNA samples from 78 children with CDGP (predominantly white males) and 112 control subjects. DNA fragments with a WAVE pattern deviant from wild type were directly sequenced. A STAT3 luciferase reporter assay in human embryonic kidney (HEK293) cells transiently transfected with the leptin receptor was used to test activity of mutant leptin. RESULTS: One child with CDGP was identified to be heterozygous for a novel missense variant (c.68C>G), which results in a proline to arginine substitution (p.P23R). This sequence variant was not identified in any of the other control subjects, but was identified in his mother who shared a similar phenotype of slim body habitus, reduced appetite and pubertal delay (menarche aged 15 years). The leptin variant showed similar stability in serum compared with wild type and did not demonstrate increased activity in an in vitro reporter gene assay. CONCLUSIONS: This is the first report of a sequence variant within the LEP gene associated with reduced body mass index rather than obesity. We hypothesize that this variant has increased bioactivity in vivo.
Asunto(s)
Apetito/genética , Leptina/genética , Pubertad Tardía/etiología , Pubertad Tardía/genética , Adolescente , Índice de Masa Corporal , Femenino , Humanos , Leptina/sangre , Masculino , Linaje , Pubertad Tardía/sangreRESUMEN
The link between obesity and diabetes is not fully understood but there is evidence to suggest that hypothalamic signalling pathways may be involved. The hypothalamic neuropeptides, pro-opiomelanocortin (POMC), neuropeptide Y (NPY) and agouti-related protein (AGRP) are central to the regulation of food intake and have been implicated in glucose homeostasis. Therefore, the expression of these genes was quantified in hypothalami from diabetic Zucker fatty (ZDF) rats and nondiabetic Zucker fatty (ZF) rats at 6, 8, 10 and 14 weeks of age. Although both strains are obese, only ZDF rats develop pancreatic degeneration and diabetes over this time period. In both ZF and ZDF rats, POMC gene expression was decreased in obese versus lean rats at all ages. By contrast, although there was the expected increase in both NPY and AGRP expression in obese 14-week-old ZF rats, the expression of NPY and AGRP was decreased in 6-week-old obese ZDF rats with hyperinsulinaemia and in 14-week-old rats with the additional hyperglycaemia. Therefore, candidate genes involved in glucose, and insulin signalling pathways were examined in obese ZDF rats over this age range. We found that expression of the ATP-sensitive potassium (K(ATP)) channel component, Kir6.2, was decreased in obese ZDF rats and was lower compared to ZF rats in each age group tested. Furthermore, immunofluorescence analysis showed that Kir6.2 protein expression was reduced in the dorsomedial and ventromedial hypothalamic nuclei of 6-week-old prediabetic ZDF rats compared to ZF rats. The Kir6.2 immunofluorescence colocalised with NPY throughout the hypothalamus. The differences in Kir6.2 expression in ZF and ZDF rats mimic those of NPY and AGRP, which could infer that the changes occur in the same neurones. Overall, these data suggest that chronic changes in hypothalamic Kir6.2 expression may be associated with the development of hyperinsulinaemia and hyperglycaemia in ZDF rats.
Asunto(s)
Proteína Relacionada con Agouti/biosíntesis , Diabetes Mellitus/metabolismo , Hipotálamo/metabolismo , Neuropéptido Y/biosíntesis , Obesidad/metabolismo , Canales de Potasio de Rectificación Interna/biosíntesis , Animales , Diabetes Mellitus/genética , Diabetes Mellitus/patología , Expresión Génica/fisiología , Glucosa/fisiología , Hiperglucemia/sangre , Hiperglucemia/genética , Hiperinsulinismo/sangre , Hiperinsulinismo/genética , Hipotálamo/crecimiento & desarrollo , Hipotálamo/patología , Inmunohistoquímica , Inflamación/patología , Insulina/fisiología , Leptina/fisiología , Masculino , Neuropéptido Y/genética , Obesidad/genética , Páncreas/patología , Canales de Potasio de Rectificación Interna/genética , Proopiomelanocortina/biosíntesis , Ratas , Ratas Wistar , Ratas Zucker , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Interactions between pro-opiomelanocortin (POMC)-derived peptides, agouti-related protein (AGRP) and the melanocortin-4 receptor (MC4-R) are central to energy homeostasis. In this study we have undertaken comprehensive pharmacological analysis of these interactions using a CHOK1 cell line stably transfected with human MC4-R. Our main objectives were (1) to compare the relative affinities and potencies of POMC-derived peptides endogenously secreted within the hypothalamus, (2) to investigate the potency of AGRP(83-132) antagonism with respect to each POMC-derived peptide and (3) to determine whether AGRP(83-132) and POMC-derived peptides act allosterically or orthosterically. We have found that beta melanocyte-stimulating hormone (betaMSH), desacetyl alpha MSH (da-alphaMSH) and adrenocorticotrophic hormone all have very similar affinities and potencies at the MC4-R compared with the presumed natural ligand, alphaMSH. Moreover, even MSH precursors, such as beta lipotrophic hormone, showed significant binding and functional activity. Therefore, many POMC-derived peptides could have important roles in appetite regulation and it seems unlikely that alphaMSH is the sole physiological ligand. We have shown that AGRP(83-132) acts as a competitive antagonist. There was no significant difference in the potency of inhibition by AGRP(83-132) or agouti(87-132) at the MC4-R, regardless of which POMC peptide was used as an agonist. Furthermore, we have found that AGRP(83-132) has no effect on the dissociation kinetics of radiolabelled Nle4,D-Phe7 MSH from the MC4-R, indicating an absence of allosteric effects. This provides strong pharmacological evidence that AGRP(83-132) acts orthosterically at the MC4-R to inhibit Gs-coupled accumulation of intracellular cAMP.
Asunto(s)
Regulación del Apetito , Fragmentos de Péptidos/farmacología , Proopiomelanocortina/metabolismo , Receptor de Melanocortina Tipo 4/antagonistas & inhibidores , alfa-MSH/análogos & derivados , Proteína Relacionada con Agouti , Animales , Unión Competitiva , Células CHO , Cricetinae , AMP Cíclico/metabolismo , Humanos , Unión Proteica , Receptor de Melanocortina Tipo 4/genética , Transfección , alfa-MSH/metabolismoRESUMEN
Bioactive peptides derived from the prohormone, pro-opiomelanocortin (POMC), are generated in neurons of the hypothalamus and act as endogenous ligands for the melanocortin-4 receptor (MC4R), a key molecule underlying appetite control and energy homeostasis. It is therefore important to understand many aspects of POMC gene regulation in the brain, as pharmacological manipulation of POMC expression/processing could be a potential strategy to combat obesity. Most studies that have analysed POMC gene expression in the hypothalamus have focused on gene transcription experiments. Ultimately, however, factors that regulate post-translational processing and secretion of peptides will have most bearing on melanocortin signalling. This article focuses on (a) current evidence that POMC is involved in obesity, (b) how POMC transcription is regulated in the hypothalamus, (c) the mechanism by which proteolytic processing of POMC is controlled in the hypothalamus and what peptides are produced and (d) which POMC-derived peptides are the most potent ligands at the melanocortin receptor in vitro and in vivo. It seems that post-translational cleavage of POMC in the hypothalamus may be regulated with respect to energy requirement. We predict that further research into hypothalamic POMC processing, and the proteolytic enzymes involved, may yield important new clues on how flux through the MC4R pathway is regulated.
Asunto(s)
Hipotálamo/metabolismo , Obesidad/metabolismo , Proopiomelanocortina/metabolismo , Transducción de Señal/fisiología , alfa-MSH/metabolismo , Animales , Apetito/genética , Predisposición Genética a la Enfermedad , Humanos , Mutación , Proopiomelanocortina/genética , Receptor de Melanocortina Tipo 4 , Receptores de Péptidos/metabolismoRESUMEN
Genetic analysis of a mouse model of major histocompatability complex (MHC)-associated autoimmune type 1 (insulin-dependent) diabetes mellitus (IDDM) has shown that the disease is caused by a combination of a major effect at the MHC and at least ten other susceptibility loci elsewhere in the genome. A genome-wide scan of 93 affected sibpair families (ASP) from the UK (UK93) indicated a similar genetic basis for human type 1 diabetes, with the major genetic component at the MHC locus (IDDM1) explaining 34% of the familial clustering of the disease (lambda(s)=2.5; refs 3,4). In the present report, we have analysed a further 263 multiplex families from the same population (UK263) to provide a total UK data set of 356 ASP families (UK356). Only four regions of the genome outside IDDM1/MHC, which was still the only major locus detected, were not excluded at lambda(s)=3 and lod=-2, of which two showed evidence of linkage: chromosome 10p13-p11 (maximum lod score (MLS)=4.7, P=3x10(-6), lambda(s)=1.56) and chromosome 16q22-16q24 (MLS=3.4, P=6.5x10(-5), lambda(s)=1.6). These and other novel regions, including chromosome 14q12-q21 and chromosome 19p13-19q13, could potentially harbour disease loci but confirmation and fine mapping cannot be pursued effectively using conventional linkage analysis. Instead, more powerful linkage disequilibrium-based and haplotype mapping approaches must be used; such data is already emerging for several type 1 diabetes loci detected initially by linkage.
Asunto(s)
Diabetes Mellitus Tipo 1/genética , Adolescente , Adulto , Mapeo Cromosómico , Predisposición Genética a la Enfermedad , Humanos , Reino UnidoRESUMEN
Susceptibility to autoimmune insulin-dependent (type 1) diabetes mellitus is determined by a combination of environmental and genetic factors, which include variation in MHC genes on chromosome 6p21 (IDDM1) and the insulin gene on chromosome 11p15 (IDDM2). However, linkage to IDDM1 and IDDM2 cannot explain the clustering of type 1 diabetes in families, and a role for other genes is inferred. In the present report we describe linkage and association of type 1 diabetes to the CTLA-4 gene (cytotoxic T lymphocyte associated-4) on chromosome 2q33 (designated IDDM12). CTLA-4 is a strong candidate gene for T cell-mediated autoimmune disease because it encodes a T cell receptor that mediates T cell apoptosis and is a vital negative regulator of T cell activation. In addition, we provide supporting evidence that CTLA-4 is associated with susceptibility to Graves' disease, another organ-specific autoimmune disease.
Asunto(s)
Antígenos de Diferenciación/genética , Cromosomas Humanos Par 2/genética , Diabetes Mellitus Tipo 1/genética , Ligamiento Genético , Inmunoconjugados , Abatacept , Antígenos CD , Antígeno CTLA-4 , Estudios de Casos y Controles , Exones/genética , Predisposición Genética a la Enfermedad , Enfermedad de Graves/genética , Humanos , Núcleo Familiar , Mutación Puntual , Polimorfismo GenéticoRESUMEN
Recent evidence suggests that a mutation of the glucagon receptor (GCG-R) gene is involved in the development of type 2 diabetes in French patients. We have examined patients from three geographically distinct regions in the UK and found the GGT40 (Gly) to AGT40 (Ser) mutation to be present in 15/691 (2.2%) of patients with type 2 (non-insulin dependent) diabetes and 1/425 (0.2%) of geographically matched controls and have therefore replicated association of the GCG-R mutation with classical type 2 diabetes (Fisher's exact test p = 0.008). An increased frequency of the mutation of the GCG-R gene was also found in probands of type 1 (insulin dependent) diabetic multiplex (affected sib pair) families, (10/404, 2.5%). However, a lack of preferential transmission from parents heterozygous for the mutation, to affected type 1 diabetic sibs may suggest population stratification. This in turn cannot be excluded as an alternative explanation for the difference in frequency of the GCG-R gene mutation between subjects with type 2 diabetes and normal controls.
Asunto(s)
Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 2/genética , Diabetes Gestacional/genética , Efecto Fundador , Ligamiento Genético , Receptores de Glucagón/genética , Ayuno , Femenino , Heterocigoto , Humanos , Hiperglucemia/genética , Masculino , Mutación , Embarazo , Reino UnidoRESUMEN
The IDDM2 locus encoding susceptibility to type 1 diabetes was mapped previously to a 4.1-kb region spanning the insulin gene and a minisatellite or variable number of tandem repeats (VNTR) locus on human chromosome 11p15.5. By 'cross-match' haplotype analysis and linkage disequilibrium mapping, we have mapped the mutation IDDM2 to within the VNTR itself. Other polymorphisms were systematically excluded as primary disease determinants. Transmission of IDDM2 may be influenced by parent-of-origin phenomena. Although we show that the insulin gene is expressed biallelically in the adult pancreas, we present preliminary evidence that the level of transcription in vivo is correlated with allelic variation within the VNTR. Allelic variation at VNTRs may play an important general role in human disease.
Asunto(s)
Diabetes Mellitus Tipo 1/genética , Insulina/genética , Repeticiones de Minisatélite , Adulto , Alelos , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Humanos Par 11 , ADN/genética , Cartilla de ADN/genética , ADN Satélite/genética , Femenino , Expresión Génica , Impresión Genómica , Haplotipos , Humanos , Desequilibrio de Ligamiento , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
The CD3 gene region on chromosome 11q23 has been implicated in susceptibility to type 1 (insulin-dependent) diabetes mellitus. Using semi-automated fluorescence-based technology, we have undertaken association and linkage analysis of a dinucleotide microsatellite in the CD3 delta (CD3D) gene. We have also performed a large case-control analysis of a restriction fragment length polymorphism (RFLP) in the CD3 epsilon (CD3E) gene, 26 kb from CD3D. We found no evidence for the previously reported association between the 8 kb allele of the RFLP and disease in a UK dataset of 403 diabetic patients and 446 nondiabetic controls. Furthermore, the use of the transmission/disequilibrium test (TDT) showed no evidence of linkage or association to type 1 diabetes at either marker locus. We conclude that the CD3 gene region does not contribute significantly to IDDM susceptibility. We have successfully applied semi-automated, fluorescence-based technology to undertake association analysis on the CD3D microsatellite. Moreover, by analysing 94 other dinucleotide repeat markers, we conclude that fluorescence-based methodology can generally be applied to large-scale, semi-automated association studies with most microsatellite markers.
Asunto(s)
Complejo CD3/genética , Mapeo Cromosómico/métodos , Diabetes Mellitus Tipo 1/genética , Fluorescencia , Desequilibrio de Ligamiento/genética , Adolescente , Adulto , Alelos , Biomarcadores , Diabetes Mellitus Tipo 1/epidemiología , Estudios de Evaluación como Asunto , Salud de la Familia , Femenino , Haplotipos/genética , Humanos , Masculino , Métodos , Linaje , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
We have searched the human genome for genes that predispose to type 1 (insulin-dependent) diabetes mellitus using semi-automated fluorescence-based technology and linkage analysis. In addition to IDDM1 (in the major histocompatibility complex on chromosome 6p21) and IDDM2 (in the insulin gene region on chromosome 11p15), eighteen different chromosome regions showed some positive evidence of linkage to disease. Linkages to chromosomes 11q (IDDM4) and 6q (IDDM5) were confirmed by replication, and chromosome 18 may encode a fifth disease locus. There are probably no genes with large effects aside from IDDM1. Therefore polygenic inheritance is indicated, with a major locus at the major histocompatibility complex.
Asunto(s)
Mapeo Cromosómico , Diabetes Mellitus Tipo 1/genética , Adolescente , Adulto , Cromosomas Humanos Par 11 , Cromosomas Humanos Par 6 , Ligamiento Genético , Marcadores Genéticos , Predisposición Genética a la Enfermedad , Genoma Humano , Humanos , Complejo Mayor de HistocompatibilidadRESUMEN
To facilitate large-scale genetic mapping of the human genome, we have developed chromosome-specific sets of microsatellite marker loci suitable for use with a fluorescence-based automated DNA fragment analyser. We present 254 dinucleotide repeat marker loci (80% from the Généthon genetic linkage map) arranged into 39 sets, covering all 22 autosomes and the X chromosome. The average distance between adjacent markers is 13 centiMorgans, and less than 4% of the genome lies more than 20 cM from the nearest marker. Each set of microsatellites consists of up to nine marker loci, with allele size ranges that do not overlap. We selected marker loci on the basis of their reliability in the polymerase chain reaction, polymorphism content, map position and the accuracy with which alleles can be scored automatically by the Genotyper program.
Asunto(s)
Mapeo Cromosómico/métodos , Sondas de ADN , ADN Satélite , Colorantes Fluorescentes , Marcadores Genéticos , Genoma Humano , Polimorfismo Genético , Secuencias Repetitivas de Ácidos Nucleicos , Automatización , Secuencia de Bases , Cromosomas Humanos , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Programas InformáticosAsunto(s)
Enfermedades Autoinmunes/fisiopatología , Diabetes Mellitus Experimental/fisiopatología , Diabetes Mellitus Tipo 1/fisiopatología , Ratones Endogámicos NOD/genética , Animales , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Mapeo Cromosómico , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Susceptibilidad a Enfermedades/inmunología , Genes , Predisposición Genética a la Enfermedad , Antígenos HLA/genética , Antígenos HLA/inmunología , Humanos , Ratones , Ratones Endogámicos C57BL/genética , Ratones Endogámicos C57BL/inmunología , Ratones Endogámicos NOD/inmunologíaRESUMEN
Type 1 or insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease of the insulin-producing pancreatic beta-cells which is determined by both genetic and environmental factors. The major histocompatibility complex and the insulin gene region (INS) on human chromosomes 6p and 11p, respectively, contain susceptibility genes. Using a mostly French data set, evidence for linkage of INS to IDDM was recently obtained but only in male meioses (suggesting involvement of maternal imprinting) and only in HLA-DR4-positive diabetics. In contrast, we find evidence for linkage in both male and female meioses and that the effect of the susceptibility gene(s) in the INS region is not dependent on the presence of HLA-DR4.