RESUMO
The helix-loop-helix (HLH) protein NEUROD1 (also known as BETA2) functions as a regulatory switch for endocrine pancreatic development. In mice homozygous for a targeted disruption of Neurod, pancreatic islet morphogenesis is abnormal and overt diabetes develops due in part to inadequate expression of the insulin gene (Ins2). NEUROD1, following its heterodimerization with the ubiquitous HLH protein E47, regulates insulin gene (INS) expression by binding to a critical E-box motif on the INS promoter. Here we describe two mutations in NEUROD1, which are associated with the development of type 2 diabetes in the heterozygous state. The first, a missense mutation at Arg 111 in the DNA-binding domain, abolishes E-box binding activity of NEUROD1. The second mutation gives rise to a truncated polypeptide lacking the carboxy-terminal trans-activation domain, a region that associates with the co-activators CBP and p300 (refs 3,4). The clinical profile of patients with the truncated NEUROD1 polypeptide is more severe than that of patients with the Arg 111 mutation. Our findings suggest that deficient binding of NEUROD1 or binding of a transcriptionally inactive NEUROD1 polypeptide to target promoters in pancreatic islets leads to the development of type 2 diabetes in humans.
Assuntos
Proteínas de Ligação a DNA/genética , Diabetes Mellitus Tipo 2/genética , Mutação/genética , Transativadores/genética , Adolescente , Adulto , Sequência de Aminoácidos , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , DNA/genética , DNA/metabolismo , Análise Mutacional de DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Feminino , Regulação da Expressão Gênica , Heterozigoto , Humanos , Insulina/genética , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Linhagem , Polimorfismo Genético/genética , Elementos de Resposta/genética , Deleção de Sequência/genética , Transativadores/química , Transativadores/metabolismo , Células Tumorais CultivadasRESUMO
The homeobox protein STF-1 appears to function as a master control switch for expression of the pancreatic program during development. Here we characterize a composite enhancer which directs STF-1 expression to pancreatic islet cells via two functional elements that recognize the nuclear factors HNF-3beta and BETA-2. In keeping with their inhibitory effects on islet cell maturation, glucocorticoids were found to repress STF-1 gene expression by interfering with HNF-3beta activity on the islet-specific enhancer. Overexpression of HNF-3beta suppressed glucocorticoid receptor-mediated inhibition of the STF-1 gene, and our results suggest that the expansion of pancreatic islet precursor cells during development may be restricted by hormonal cues which regulate STF-1 gene expression.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Sequências Hélice-Alça-Hélice , Proteínas de Homeodomínio/genética , Ilhotas Pancreáticas/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Northern Blotting , Western Blotting , Células COS , Pegada de DNA , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Glucocorticoides/farmacologia , Células HeLa , Fator 3-beta Nuclear de Hepatócito , Proteínas de Homeodomínio/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Fatores de Transcrição/genética , TransfecçãoRESUMO
The cyclic AMP (cAMP)-responsive factor CREB promotes cellular gene expression, following its phosphorylation at Ser133, via recruitment of the coactivator paralogs CREB-binding protein (CBP) and p300. CBP and p300, in turn, appear to mediate target gene induction via their association with RNA polymerase II complexes and via intrinsic histone acetyltransferase activities that mobilize promoter-bound nucleosomes. In addition to cAMP, a wide variety of stimuli, including hypoxia, UV irradiation, and growth factor addition, induce Ser133 phosphorylation with stoichiometry and kinetics comparable to those induced by cAMP. Yet a number of these signals are incapable of promoting target gene activation via CREB phosphorylation per se, suggesting the presence of additional regulatory events either at the level of CREB-CBP complex formation or in the subsequent recruitment of the transcriptional apparatus. Here we characterize a Tyr134Phe CREB mutant that behaves as a constitutive activator in vivo. Like protein kinase A (PKA)-stimulated wild-type CREB, the Tyr134Phe polypeptide was found to stimulate target gene expression via the Ser133-dependent recruitment of CBP and p300. Biochemical studies reveal that mutation of Tyr134 to Phe lowers the K(m) for PKA phosphorylation and thereby induces high levels of constitutive Ser133 phosphorylation in vivo. Consistent with its constitutive activity, Tyr134Phe CREB strongly promoted differentiation of PC12 cells in concert with suboptimal doses of nerve growth factor. Taken together, these results demonstrate that Ser133 phosphorylation is sufficient for cellular gene activation and that additional signal-dependent modifications of CBP or p300 are not required for recruitment of the transcriptional apparatus to the promoter.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Regulação da Expressão Gênica , Ativação Transcricional , Animais , Diferenciação Celular/genética , Mutação , Células PC12 , Regiões Promotoras Genéticas , Ratos , Transdução de Sinais/genéticaRESUMO
The present studies examined the effects of chronic interleukin-1 (IL-1 beta) infusion on glucose homeostasis and insulin secretion in male Sprague Dawley rats. IL-1 beta (4 micrograms per day) or saline was infused over a six day period using mini-osmotic pumps, surgically inserted under light ether anesthesia. Saline-infused rats were fed the amount of food consumed by their respective pair in the IL-1 beta group on the previous day. IL-1 beta infusion resulted in decreased food intake and significant body weight loss as well as increased liver and kidney weights. IL-1 beta infusion resulted in fasting hypoglycemia as well as elevated blood glucose levels in response to an oral glucose load compared to controls. Glucose-induced insulin secretion from the isolated perfused pancreas was significantly lower in IL-1 beta treated rats compared to controls. These data demonstrate that chronic IL-1 beta administration alters glucose homeostasis and impairs glucose-induced insulin secretion.
Assuntos
Glicemia/análise , Insulina/metabolismo , Interleucina-1/farmacologia , Animais , Peso Corporal/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Jejum , Glucose/farmacologia , Homeostase/efeitos dos fármacos , Hidrocortisona/sangue , Insulina/sangue , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , Masculino , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Triglicerídeos/sangueRESUMO
Glutamine catabolism in mammalian liver is catalysed by a unique isoenzyme of phosphate-activated glutaminase. The full coding and 5' untranslated sequence for rat hepatic glutaminase was isolated by screening lambda ZAP cDNA libraries and a Charon 4a rat genomic library. The sequence produces a mRNA 2225 nt in length, encoding a polypeptide of 535 amino acid residues with a calculated molecular mass of 59.2 kDa. The deduced amino acid sequence of rat liver glutaminase shows 86% similarity to that of rat kidney glutaminase and 65% similarity to a putative glutaminase from Caenorhabditis elegans. A genomic clone to rat liver glutaminase was isolated that contains 3.5 kb of the gene and 7.5 kb of the 5' flanking region. The 1 kb immediately upstream of the hepatic glutaminase gene (from -1022 to +48) showed functional promoter activity in HepG2 hepatoma cells. This promoter region did not respond to treatment with cAMP, but was highly responsive (10-fold stimulation) to the synthetic glucocorticoid dexamethasone. Subsequent 5' deletion analysis indicated that the promoter region between -103 and +48 was sufficient for basal promoter activity. This region does not contain an identifiable TATA element, indicating that transcription of the glutaminase gene is driven by a TATA-less promoter. The region responsive to glucocorticoids was mapped to -252 to -103 relative to the transcription start site.
Assuntos
Glutaminase/genética , Fígado/enzimologia , Regiões Promotoras Genéticas , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Caenorhabditis elegans/enzimologia , Cloranfenicol O-Acetiltransferase/biossíntese , DNA Complementar , Éxons , Biblioteca Genômica , Glucocorticoides/farmacologia , Glutaminase/biossíntese , Glutaminase/química , Rim/enzimologia , Dados de Sequência Molecular , RNA Mensageiro/biossíntese , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Homologia de Sequência de Aminoácidos , Transcrição Gênica/efeitos dos fármacosRESUMO
When mammals fast, glucose homeostasis is achieved by triggering expression of gluconeogenic genes in response to glucagon and glucocorticoids. The pathways act synergistically to induce gluconeogenesis (glucose synthesis), although the underlying mechanism has not been determined. Here we show that mice carrying a targeted disruption of the cyclic AMP (cAMP) response element binding (CREB) protein gene, or overexpressing a dominant-negative CREB inhibitor, exhibit fasting hypoglycaemia [corrected] and reduced expression of gluconeogenic enzymes. CREB was found to induce expression of the gluconeogenic programme through the nuclear receptor coactivator PGC-1, which is shown here to be a direct target for CREB regulation in vivo. Overexpression of PGC-1 in CREB-deficient mice restored glucose homeostasis and rescued expression of gluconeogenic genes. In transient assays, PGC-1 potentiated glucocorticoid induction of the gene for phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme in gluconeogenesis. PGC-1 promotes cooperativity between cyclic AMP and glucocorticoid signalling pathways during hepatic gluconeogenesis. Fasting hyperglycaemia is strongly correlated with type II diabetes, so our results suggest that the activation of PGC-1 by CREB in liver contributes importantly to the pathogenesis of this disease.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Gluconeogênese , Fígado/metabolismo , Fatores de Transcrição/fisiologia , Adenoviridae/genética , Animais , Glicemia/metabolismo , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/genética , Jejum , Regulação Enzimológica da Expressão Gênica , Humanos , Hiperglicemia/sangue , Hiperglicemia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Ser-133 phosphorylation of CREB within the kinase-inducible domain (KID) promotes target gene activation via complex formation with the KIX domain of the coactivator CBP. Concurrent phosphorylation of CREB at Ser-142 inhibits transcriptional induction via an unknown mechanism. Unstructured in the free state, KID folds into a helical structure upon binding to KIX. Using site-directed mutagenesis based on the NMR structure of the KID:KIX complex, we have examined the mechanisms by which Ser-133 and Ser-142 phosphorylation regulate CREB activity. Our results indicate that phospho-Ser-133 stablizes whereas phospho-Ser-142 disrupts secondary structure-mediated interactions between CREB and CBP. Thus, differential phosphorylation of CREB may form the basis by which upstream signals regulate the specificity of target gene activation.