RESUMO
In Type 2 diabetes (T2D), elevated lipid levels have been suggested to contribute to insulin resistance and ß-cell dysfunction. We previously reported that the expression of the PGE2 receptor EP3 is elevated in islets of T2D individuals and is preferentially stimulated by palmitate, leading to ß-cell failure. The mouse EP3 receptor generates three isoforms by alternative splicing which differ in their C-terminal domain and are referred to as mEP3α, mEP3ß, and mEP3γ. We bring evidence that the expression of the mEP3γ isoform is elevated in islets of diabetic db/db mice and is selectively upregulated by palmitate. Specific knockdown of the mEP3γ isoform restores the expression of ß-cell-specific genes and rescues MIN6 cells from palmitate-induced dysfunction and apoptosis. This study indicates that palmitate stimulates the expression of the mEP3γ by a posttranscriptional mechanism, compared to the other spliced isoforms, and that the de novo synthesized ceramide plays an important role in FFA-induced mEP3γ expression in ß-cells. Moreover, induced levels of mEP3γ mRNA by palmitate or ceramide depend on p38 MAPK activation. Our findings suggest that mEP3γ gene expression is regulated at the posttranscriptional level and defines the EP3 signaling axis as an important pathway mediating ß-cell-impaired function and demise.
Assuntos
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Camundongos , Animais , Receptores de Prostaglandina E/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Palmitatos/metabolismo , Ceramidas/metabolismo , Receptores de Prostaglandina E Subtipo EP3/genética , Receptores de Prostaglandina E Subtipo EP3/metabolismoRESUMO
NF-κB is a well-characterized transcription factor, widely known for its roles in inflammation and immune responses, as well as in control of cell division and apoptosis. However, its function in ß-cells is still being debated, as it appears to depend on the timing and kinetics of its activation. To elucidate the temporal role of NF-κB in vivo, we have generated two transgenic mouse models, the ToIß and NOD/ToIß mice, in which NF-κB activation is specifically and conditionally inhibited in ß-cells. In this study, we present a novel function of the canonical NF-κB pathway during murine islet ß-cell development. Interestingly, inhibiting the NF-κB pathway in ß-cells during embryogenesis, but not after birth, in both ToIß and NOD/ToIß mice, increased ß-cell turnover, ultimately resulting in a reduced ß-cell mass. On the NOD background, this was associated with a marked increase in insulitis and diabetes incidence. While a robust nuclear immunoreactivity of the NF-κB p65-subunit was found in neonatal ß-cells, significant activation was not detected in ß-cells of either adult NOD/ToIß mice or in the pancreata of recently diagnosed adult T1D patients. Moreover, in NOD/ToIß mice, inhibiting NF-κB post-weaning had no effect on the development of diabetes or ß-cell dysfunction. In conclusion, our data point to NF-κB as an important component of the physiological regulatory circuit that controls the balance of ß-cell proliferation and apoptosis in the early developmental stages of insulin-producing cells, thus modulating ß-cell mass and the development of diabetes in the mouse model of T1D.
RESUMO
Elevated levels of lipids, in particular saturated fatty acids, are known to be associated with type 2 diabetes (T2D) and to have a negative effect on ß-cell function and survival. We bring new evidence indicating that palmitate up-regulates cyclooxygenase-2 (COX-2) expression levels in human islets and in MIN6 ß cells, and that it is elevated in islets isolated from T2D donors. Both small interfering specific cyclooxygenase-2 small interfering RNA (siRNA) or the COX-2 inhibitor celecoxib significantly inhibited apoptosis induced by palmitate. Prostaglandin E2 (PGE2), the predominant product of COX-2 enzymatic activity, activates membrane receptors, which are members of the GPCR-family (EP1-EP4). In the present study, elevated expression of the PGE2 receptor subtype 3 (EP3) receptor was observed in ß cells exposed to palmitate and in islets from individuals with T2D. Down-regulation of the pathway using EP3 siRNA or the specific L-798,106 antagonist markedly decreased the levels of palmitate-induced apoptosis. Altogether, our data put forward the COX-2-PGE2-EP3 pathway as one of the mediators of palmitate-induced apoptosis in ß-cells.-Amior, L., Srivastava, R., Nano, R., Bertuzzi, F., Melloul, D. The role of Cox-2 and prostaglandin E2 receptor EP3 in pancreatic ß-cell death.
Assuntos
Ciclo-Oxigenase 2/metabolismo , Células Secretoras de Insulina/metabolismo , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP3/metabolismo , Celecoxib/farmacologia , Linhagem Celular , Ciclo-Oxigenase 2/genética , Inibidores de Ciclo-Oxigenase 2/farmacologia , Feminino , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Palmitatos/farmacologia , Receptores de Prostaglandina E Subtipo EP2/genética , Receptores de Prostaglandina E Subtipo EP3/genéticaRESUMO
Cystic fibrosis (CF)-related diabetes (CFRD) has become a critical complication that seriously affects the clinical outcomes of CF patients. Although CFRD has emerged as the most common nonpulmonary complication of CF, little is known about its etiopathogenesis. Additionally, whether oxidative stress (OxS), a common feature of CF and diabetes, influences CFRD pathophysiology requires clarification. The main objective of this study was to shed light on the role of the cystic fibrosis transmembrane conductance regulator (CFTR) in combination with OxS in insulin secretion from pancreatic ß-cells. CFTR silencing was accomplished in MIN6 cells by stable expression of small hairpin RNAs (shRNA), and glucose-induced insulin secretion was evaluated in the presence and absence of the valuable prooxidant system iron/ascorbate (Fe/Asc; 0.075/0.75 mM) along with or without the antioxidant Trolox (1 mM). Insulin output from CFTR-silenced MIN6 cells was significantly reduced (â¼ 70%) at basal and at different glucose concentrations compared with control Mock cells. Furthermore, CFTR silencing rendered MIN6 cells more sensitive to OxS as evidenced by both increased lipid peroxides and weakened antioxidant defense, especially following incubation with Fe/Asc. The decreased insulin secretion in CFTR-silenced MIN6 cells was associated with high levels of NF-κB (the major participant in inflammatory responses), raised apoptosis, and diminished ATP production in response to the Fe/Asc challenge. However, these defects were alleviated by the addition of Trolox, thereby pointing out the role of OxS in aggravating the effects of CFTR deficiency. Our findings indicate that CFTR deficiency in combination with OxS may contribute to endocrine cell dysfunction and insulin secretion, which at least in part may explain the development of CFRD.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Estresse Oxidativo/genética , RNA Mensageiro/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Western Blotting , Catalase/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Cromanos/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Técnicas de Silenciamento de Genes , Inativação Gênica , Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Células HEK293 , Humanos , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Ferro/farmacologia , Peroxidação de Lipídeos/genética , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Superóxido Dismutase/metabolismo , Oligoelementos/farmacologiaRESUMO
Pancreatic islet transplantation, a treatment for type 1 diabetes, has met significant challenges, as a substantial fraction of the islet mass fails to engraft, partly due to death by apoptosis in the peri- and post-transplantation periods. Previous evidence has suggested that NF-κB activation is involved in cytokine-mediated ß-cell apoptosis and regulates the expression of pro-inflammatory and chemokine genes. We therefore sought to explore the effects of ß-cell-specific inhibition of NF-κB activation as a means of cytoprotection in an allogeneic model of islet transplantation. To this end, we used islets isolated from the ToI-ß transgenic mouse, where NF-κB signalling can specifically and conditionally be inhibited in ß-cells by expressing an inducible and non-degradable form of IκBα regulated by the tet-on system. Our results show that ß-cell-specific blockade of NF-κB led to a prolonged islet graft survival, with a relative higher preservation of the engrafted endocrine tissue and reduced inflammation. Importantly, a longer delay in allograft rejection was achieved when mice were systemically treated with the proteasome inhibitor, Bortezomib. Our findings emphasize the contribution of NF-κB activation in the allograft rejection process, and suggest an involvement of the CXCL10/IP-10 chemokine. Furthermore, we suggest a potential, readily available therapeutic agent that may temper this process.
Assuntos
Sobrevivência de Enxerto , Células Secretoras de Insulina/metabolismo , Transplante das Ilhotas Pancreáticas , NF-kappa B/antagonistas & inibidores , Animais , Quimiocina CXCL10/metabolismo , Ativação Enzimática , Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/metabolismo , Sobrevivência de Enxerto/efeitos dos fármacos , Sobrevivência de Enxerto/genética , Antígenos H-2/genética , Células Secretoras de Insulina/efeitos dos fármacos , Interferon gama/farmacologia , Interleucina-1beta/farmacologia , Camundongos , Camundongos Transgênicos , Nitritos/metabolismo , Complexo de Endopeptidases do Proteassoma , Inibidores de Proteínas Quinases/farmacologia , Transplante HomólogoRESUMO
Insulin-producing pancreatic ß cells are functionally impaired or destroyed in diabetes mellitus. The onset of type 1 diabetes (T1D) represents the culmination of a prolonged prediabetic phase of immune-mediated ß-cell destruction. To assess the in vivo metabolic status of these cells, we used the ATP-sensitive firefly luciferase bioluminescence imaging approach, as a noninvasive probe to monitor pathological alterations in ß-cell function in the nonobese-diabetic (NOD) mouse model of T1D. Hence, we generated the ToIß-NOD transgenic mice in which doxycycline-inducible luciferase gene is selectively expressed in ß cells. A sharp reduction in bioluminescence emitted in vivo from ß cells at the early stages, preceded by several weeks of a limited reduction in ß-cell mass. Since this decline could be due to the ongoing inflammatory process occurring in vivo, we exposed control islets to inflammatory cytokines and observed a dramatic decrease in luciferase luminescence, which appears to be due in part to a decrease in protein levels and a drop in intracellular ATP levels. This is the first evidence that selective expression of the luciferase gene represents a sensitive method for noninvasive in vivo monitoring of early ß-cell dysfunction, subtle metabolic changes, such as endogenous ATP levels, indicative of a pathological condition in a tissue at the cellular level.
Assuntos
Células Secretoras de Insulina/metabolismo , Luciferases/metabolismo , Medições Luminescentes/métodos , Envelhecimento , Animais , Glucose/farmacologia , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Luciferases/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Fatores de TempoRESUMO
Type 1 diabetes is characterized by the infiltration of inflammatory cells into pancreatic islets of Langerhans, followed by the selective and progressive destruction of insulin-secreting beta-cells. Islet infiltrating leukocytes secrete cytokines including IL-1beta and IFN-gamma, which contribute to beta-cell death. In vitro evidence suggests that cytokine-induced activation of the transcription factor NF-kappaB is an important component of the signal triggering beta-cell apoptosis. To study the role of NF-kappaB in vivo we generated a transgenic mouse line expressing a degradation-resistant NF-kappaB protein inhibitor (DeltaNIkappaBalpha) and the luciferase gene, acting specifically in beta-cells, in an inducible and reversible manner, by using the tet-on regulation system. Using this new mouse model, termed the ToI-beta mouse (for Tet-Ondelta I kappaB in beta-cells) we have previously shown in vitro, that islets expressing the DeltaNIkappaBalpha protein were resistant to the deleterious effects of IL-1beta and IFN-gamma, as assessed by reduced NO production and beta-cell apoptosis. In vivo, a nearly complete protection against multiple low dose streptozocin-induced diabetes was observed, with reduced intra-islet lymphocytic infiltration. In the present study we demonstrate the tight regulated and reversible expression of the DeltaNIkappaBalpha transgene in the ToI-beta mouse model as well as the effect of its overexpression on glucose metabolism and insulin secretion. The results show a lack of effect of transgene induction on both in vivo glucose tolerance tests and in vitro islet insulin secretion and content. Furthermore, to prove the tight control of induction in the model, luciferase mediated light emission was only detected at constant levels in Dox-treated double transgenic mice or islets as well as in a model of islet transplantation. Upon removal of the inducing stimulus, complete reversal of both NF-kappaB inhibition and luciferase activity were observed. Together, our results show the ToI-beta mouse model to be a highly controlled and very accurate model for examining pancreatic beta-cell-specific temporal inhibition of NF-kappaB.
Assuntos
Células Secretoras de Insulina/fisiologia , NF-kappa B/fisiologia , Animais , Apoptose/efeitos dos fármacos , Citocinas/fisiologia , Glucose/metabolismo , Teste de Tolerância a Glucose , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/metabolismo , Interferon gama/metabolismo , Interferon gama/farmacologia , Interleucina-1beta/farmacologia , Camundongos , Camundongos Transgênicos , Modelos Animais , NF-kappa B/antagonistas & inibidoresRESUMO
Apoptotic beta-cell death appears to be central to the pathogenesis of Type 1 diabetes mellitus and in islet graft rejection. The beta-cell destruction is partially mediated by cytokines, such as IL-1beta (interleukin 1beta), TNFalpha (tumour necrosis factor alpha) and IFN-gamma (interferon gamma). IL-1beta and TNFalpha mediate activation of the transcription factor NF-kappaB (nuclear factor kappaB) pathway. Use of a degradation-resistant NF-kappaB protein inhibitor (DeltaNIkappaBalpha), specifically expressed in beta-cells, significantly reduced IL-1beta+IFN-gamma-induced apoptosis. Moreover, in vivo, it protected against multiple low-dose streptozocin-induced diabetes, with reduced intra-islet lymphocytic infiltration. Thus beta-cell-specific activation of NF-kappaB is a key event in the progressive loss of beta-cells in diabetes. Inhibition of this process could be a potential effective strategy for beta-cell protection.
Assuntos
Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , NF-kappa B/metabolismo , Animais , Morte Celular , Citocinas/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , HumanosRESUMO
KLF11 (TIEG2) is a pancreas-enriched transcription factor that has elicited significant attention because of its role as negative regulator of exocrine cell growth in vitro and in vivo. However, its functional role in the endocrine pancreas remains to be established. Here, we report, for the first time, to our knowledge, the characterization of KLF11 as a glucose-inducible regulator of the insulin gene. A combination of random oligonucleotide binding, EMSA, luciferase reporter, and chromatin immunoprecipitation assays shows that KLF11 binds to the insulin promoter and regulates its activity in beta cells. Genetic analysis of the KLF11 gene revealed two rare variants (Ala347Ser and Thr220Met) that segregate with diabetes in families with early-onset type 2 diabetes, and significantly impair its transcriptional activity. In addition, analysis of 1,696 type 2 diabetes mellitus and 1,776 normoglycemic subjects show a frequent polymorphic Gln62Arg variant that significantly associates with type 2 diabetes mellitus in North European populations (OR = 1.29, P = 0.00033). Moreover, this variant alters the corepressor mSin3A-binding activity of KLF11, impairs the activation of the insulin promoter and shows lower levels of insulin expression in pancreatic beta cells. In addition, subjects carrying the Gln62Arg allele show decreased plasma insulin after an oral glucose challenge. Interestingly, all three nonsynonymous KLF11 variants show increased repression of the catalase 1 promoter, suggesting a role in free radical clearance that may render beta cells more sensitive to oxidative stress. Thus, both functional and genetic analyses reveal that KLF11 plays a role in the regulation of pancreatic beta cell physiology, and its variants may contribute to the development of diabetes.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Diabetes Mellitus Tipo 2/genética , Regulação da Expressão Gênica , Insulina/metabolismo , Ilhotas Pancreáticas/fisiologia , Polimorfismo Genético , Proteínas Repressoras/fisiologia , Fatores de Transcrição/fisiologia , Proteínas Reguladoras de Apoptose , Composição de Bases , Sequência de Bases , Estudos de Casos e Controles , Proteínas de Ciclo Celular/genética , Imunoprecipitação da Cromatina , Europa (Continente) , Componentes do Gene , Humanos , Insulina/genética , Luciferases , Dados de Sequência Molecular , Linhagem , Regiões Promotoras Genéticas/genética , Proteínas Repressoras/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Fatores de Transcrição/genéticaRESUMO
Differentiation of early foregut endoderm into pancreatic endocrine and exocrine cells depends on a cascade of gene activation events controlled by various transcription factors. The first molecular marker identified that specifies the early pancreatic epithelium is the homeodomain-containing transcription factor PDX-1. Its absence in mice and humans during development leads to agenesis of the pancreas. Later, it becomes restricted primarily to beta cells where it regulates the expression of beta cell-specific genes, and, most importantly, mediates the glucose effect on insulin gene transcription. Although exposure of beta cells to high glucose concentrations for relatively short periods stimulates insulin gene expression, chronic exposure has adverse effects on many beta-cell functions, including insulin gene transcription. These events appear to correlate with pdx-1 gene expression and its ability to bind the insulin gene. We consider that loss of PDX-1 function or altered pdx-1 gene expression due to mutations or functional impairment of transcription factors controlling its expression can lead to diabetes.
Assuntos
Proteínas de Homeodomínio , Ilhotas Pancreáticas/crescimento & desenvolvimento , Ilhotas Pancreáticas/fisiologia , Transativadores/fisiologia , Animais , HumanosRESUMO
Transcriptional coregulators modulate the activity of transcription factors and are required for the proper regulation of gene expression. One transcriptional coactivator, peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha), plays an important role in the control of energy metabolism and has been associated with type 2 diabetes. A recent paper by Fan et al. provides new information about the posttranslational regulation of PGC-1alpha activity. This Perspective discusses the implications of these findings with respect to diabetes and aging.
Assuntos
Fatores de Transcrição/fisiologia , Animais , Humanos , Processamento de Proteína Pós-Traducional , Fatores de Transcrição/biossíntese , Fatores de Transcrição/metabolismoRESUMO
Neonatal diabetes mellitus can be transient or permanent. The severe form of permanent neonatal diabetes mellitus can be associated with pancreas agenesis. Normal pancreas development is controlled by a cascade of transcription factors, where insulin promoter factor 1 (IPF1) plays a crucial role. Here, we describe two novel mutations in the IPF1 gene leading to pancreas agenesis. Direct sequence analysis of exons 1 and 2 of the IPF1 gene revealed two point mutations within the homeobox in exon 2. Genetic analysis of the parents showed that each mutation was inherited from one parent. Mutations localized in helices 1 and 2, respectively, of the homeodomain, decreased the protein half-life significantly, leading to intracellular IPF1 levels of 36% and 27% of wild-type levels. Both mutant forms of IPF1 were normally translocated to the nucleus, and their DNA binding activity on different known target promoters was similar to that of the wild-type protein. However, transcriptional activity of both mutant IPF1 proteins, alone or in combination with HNF3 beta/Foxa2, Pbx1, or the heterodimer E47-beta 2 was reduced, findings accounted for by decreased IPF1 steady state levels and not by impaired protein-protein interactions. We conclude that the IPF1 level is critical for human pancreas formation.
Assuntos
Proteínas de Homeodomínio , Mutação/fisiologia , Pâncreas/anormalidades , Pâncreas/crescimento & desenvolvimento , Transativadores/genética , Transativadores/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Células Cultivadas , Cricetinae , DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Genes Reporter/genética , Teste de Tolerância a Glucose , Proteínas de Fluorescência Verde , Meia-Vida , Humanos , Marcação por Isótopo , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Dados de Sequência Molecular , Mutagênese , Plasmídeos/genética , Testes de Precipitina , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores/química , Ativação Transcricional/genética , Transfecção , Translocação Genética/genéticaRESUMO
The homeodomain-containing transcription factor pancreatic duodenal homeobox 1 (PDX-1) plays a key role in pancreas development and in beta-cell function. Upstream sequences of the gene up to about -6 kb show islet-specific activity in transgenic mice. Attempts to identify functional regulatory elements involved in the controlled expression of the pdx-1 gene led to the identification of distinct distal beta-cell-specific enhancers in human and rat genes. Three additional sequences, conserved between the mouse and the human 5'-flanking regions, two of which are also found in the chicken gene, conferred beta-cell-specific expression on a reporter gene, albeit to different extents. A number of transcription factors binding to and modulating the transcriptional activity of the regulatory elements were identified, such as hepatocyte nuclear factor (HNF)-3beta, HNF-1alpha, SP1/3, and, interestingly, PDX-1 itself. A fourth conserved region was localized to the proximal promoter around an E-box motif and was found to bind members of the upstream stimulatory factor (USF) family of transcription factors. We postulate that disruption of pdx-1 cis-acting regulatory sequences and/or mutations or functional impairment of transcription factors controlling the expression of the gene can lead to diabetes.
Assuntos
Regulação da Expressão Gênica , Proteínas de Homeodomínio , Ilhotas Pancreáticas/fisiologia , Transativadores/genética , Animais , Sequência de Bases/genética , Sequência Conservada , Elementos Facilitadores Genéticos , Humanos , Dados de Sequência Molecular , Regiões Promotoras GenéticasRESUMO
Insulin is expressed exclusively in the adult beta-cells of the islets of Langerhans. Pancreatic Duodenum Homeobox-1 (PDX-1) is a major regulator of transcription in these cells. It transactivates the insulin gene by binding to a specific DNA motif in its promoter region. Glucose, the main physiological regulator of insulin secretion, also regulates insulin gene transcription through PDX-1. While acute exposure to high glucose concentrations causes an increase in PDX-1 binding, and consequently in insulin mRNA levels, chronic hyperglycemia (toxic to the beta-cell) leads to a decrease in PDX-1 and insulin levels. PDX-1 is absolutely required for pancreas development. In view of the selective expression in adult beta-cells, pancreatic agenesis in both the pdx-1 null mouse and a human carrying a homozygous mutation of PDX-1 was an unexpected and remarkable finding. The homozygous clinical phenotype was neonatal diabetes mellitus (DM) and exocrine insufficiency. Heterozygosity for PDX-1 mutations was found in some individuals with a newly characterized subtype of maturity-onset diabetes of the young (MODY4) and in others with type 2 DM. This review underlines the unique role of PDX-1 in maintaining adult beta-cell-specific functions in normal and disease-related states.