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1.
Transgenic Res ; 13(5): 463-73, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15587270

RESUMO

We report here knock-in of diphteria toxin A chain (dta) gene at the Ins2 locus, using the strategy previously employed to insert lacZ under control of the Ins2 promoter. Mutant Ins2(dta/+), Ins2(dta/lacZ) or Ins2(lacZ/+) mouse pups were generated by breeding and analyzed to study the effects of toxigenetic beta-cell ablation on islet development and to localize the extrapancreatic Ins2 expression site in the brain. Ins2(dta/+) and Ins2(dta/lacZ) pups developed a severe diabetic ketoacidosis and died rapidly. Histological analysis of their pancreas revealed that beta-cells completely disappeared in their islets as evidenced by loss of lacZ activity or insulin immunonostaining. beta-cell ablation did not alter the size of other islet cell populations which were normal at birth, although the glucagon-cell population was reduced by 85% at embryonic day E12.5. In the brain, comparative analysis of lacZ expression in Ins2(lacZ/+) and Ins2(dta/laZ) mice identified the choroid plexus (CP) as a major Ins2 expression site. This finding was confirmed by RT-PCR analysis of insulin transcripts in RNAs prepared from microdissected wild-type CP. Transcripts for other key beta-cell markers, with the notable exception of Pdx-1, were also found in CP RNAs. These results must revive interest in studies focused on extrapancreatic insulin gene expression.


Assuntos
Plexo Corióideo/metabolismo , Toxina Diftérica/genética , Regulação da Expressão Gênica no Desenvolvimento , Ilhotas Pancreáticas/metabolismo , Pâncreas/metabolismo , Fragmentos de Peptídeos/genética , Animais , Plexo Corióideo/patologia , Cetoacidose Diabética/metabolismo , Cetoacidose Diabética/patologia , Toxina Diftérica/metabolismo , Vetores Genéticos/genética , Insulina/metabolismo , Ilhotas Pancreáticas/patologia , Óperon Lac/genética , Camundongos , Camundongos Transgênicos , Pâncreas/patologia , Fragmentos de Peptídeos/metabolismo , Regiões Promotoras Genéticas
2.
J Immunol ; 172(1): 25-33, 2004 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-14688305

RESUMO

Deciphering mechanisms involved in failure of self tolerance to preproinsulin-2 is a key issue in type 1 diabetes. We used nonautoimmune 129SV/Pas mice lacking preproinsulin-2 to study the immune response to preproinsulin-2. In these mice, a T cell response was detected after immunization with several preproinsulin-2 peptides and confirmed by generating hybridomas. Activation of some of these hybridomas by wild-type (wt) islet cells or recombinant murine proinsulin-2 demonstrated that two epitopes can be generated from the naturally expressed protein. Although T cells from wt mice responded to preproinsulin-2 peptides, we could not detect a response to the naturally processed epitopes in these mice. Moreover, after immunization with recombinant whole proinsulin-2, a T cell response was detected in preproinsulin-2-deficient but not in wt mice. This suggests that islet preproinsulin-2-autoreactive T cells are functionally eliminated in wt mice. We used a transplantation model to evaluate the relevance of reactivity to preproinsulin-2 in vivo. Wild-type preproinsulin-2-expressing islets transplanted in preproinsulin-2-deficient mice elicited a mononuclear cell infiltration and insulin Abs. Graft infiltration was further increased by immunization with preproinsulin-2 peptides. Preproinsulin-2 expression thus shapes the immune response and prevents self reactivity to the islet. Moreover, islet preproinsulin-2 primes an immune response to preproinsulin-2 in deficient mice.


Assuntos
Regulação da Expressão Gênica/imunologia , Proinsulina/genética , Proinsulina/imunologia , Precursores de Proteínas/genética , Precursores de Proteínas/imunologia , Sequência de Aminoácidos , Animais , Apresentação de Antígeno/genética , Hibridomas , Insulina , Interleucina-2/metabolismo , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Transplante das Ilhotas Pancreáticas/imunologia , Transplante das Ilhotas Pancreáticas/patologia , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/imunologia , Proinsulina/administração & dosagem , Proinsulina/deficiência , Isoformas de Proteínas/administração & dosagem , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Precursores de Proteínas/administração & dosagem , Precursores de Proteínas/deficiência , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Tolerância ao Transplante/genética , Vacinação
3.
Int J Exp Diabesity Res ; 4(1): 7-12, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12745665

RESUMO

The authors have derived a new beta-cell line (betaIns2(-/-lacZ)) from Ins2-/- mice that carry the lacZ reporter gene under control of the Ins2 promoter. betaIns2(-/-lacZ) cells stained positively using anti-insulin antibody, expressed beta-cell-specific genes encoding the transcription factor PDX-1, glucokinase, and Glut-2, retained glucose-responsiveness for insulin secretion, and expressed the lacZ gene. Analysis of Ins1 expression by reverse transcriptase-polymerase chain reaction (RT-PCR) showed that Ins1 transcripts were significantly raised to compensate for the lack of Ins2 transcripts in betaIns2(-/-lacZ) cells, as compared to those found in betaTC1 cells expressing both Ins1/Ins2. Thus, transcriptional up-regulation of the remaining functional insulin gene in Ins2-/- mice could potentially contribute to the beta-cell adaptation exhibited by these mutants, in addition to the increase in beta-cell mass that we previously reported. We have also shown that lacZ expression, as analyzed by determining beta-galactosidase activity, was up-regulated by incubating betaIns2(-/-lacZ) cells with GLP-1 and/or IBMX, 2 known stimulators of insulin gene expression. These cells thus represent a new tool for testing of molecules capable of stimulating Ins2 promoter activity.


Assuntos
Resistência à Insulina/genética , Insulina/deficiência , Animais , Sequência de Bases , Linhagem Celular , Cruzamentos Genéticos , Primers do DNA , Glucose/farmacologia , Imuno-Histoquímica , Insulina/genética , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , beta-Galactosidase/análise , beta-Galactosidase/genética
4.
J Clin Invest ; 111(6): 851-7, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12639991

RESUMO

Accumulating evidence favors a role for proinsulin as a key autoantigen in diabetes. In the mouse, two proinsulin isoforms coexist. Most studies point to proinsulin 2 as the major isoform recognized by T cells in the NOD mouse. We studied mice in which a null proinsulin 2 mutation was transferred from proinsulin 2-deficient 129 mice onto the NOD background along with 16 genetic markers (including I-A(g7) MHC molecule) associated with diabetes. Intercross mice from the fourth backcross generation showed that proinsulin 2(-/-) mice develop accelerated insulitis and diabetes. The high prevalence of anti-insulin autoantibodies in proinsulin 2(-/-) mice indicates that diabetes acceleration relates to altered recognition of proinsulin. The prevalence of anti-glutamic acid decarboxylase autoantibodies and of sialitis is not increased in proinsulin 2(-/-) mice. We give evidence that proinsulin 2 expression leads to silencing of T cells specific for an epitope shared by proinsulin 1 and proinsulin 2. In the human, alleles located in the VNTR region flanking the insulin gene control beta cell response to glucose and proinsulin expression in the thymus and are key determinants of diabetes susceptibility. Proinsulin 2(-/-) NOD mice provide a model to study the role of thymic expression of insulin in susceptibility to diabetes.


Assuntos
Diabetes Mellitus Tipo 1/etiologia , Proinsulina/fisiologia , Transferência Adotiva , Sequência de Aminoácidos , Animais , Autoimunidade , Diabetes Mellitus Tipo 1/imunologia , Glutamato Descarboxilase/imunologia , Anticorpos Anti-Insulina/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Dados de Sequência Molecular , Proinsulina/deficiência
5.
Diabetes ; 51 Suppl 3: S489-93, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12475795

RESUMO

Insulin is a major disease determinant in type 1 diabetes, type 2 diabetes, and related disorders. The role of variations in the expression of the insulin gene has been proposed in genetic susceptibility to the three pathological conditions in humans. In contrast to humans, rodents express two proinsulin isoforms. One isoform, proinsulin 1, is expressed exclusively in islets. The second, proinsulin 2, is expressed in islets and in other tissues, especially the thymus. We took advantage of the expression of these two isoforms to introduce a null proinsulin 2 allele in NOD mice and to evaluate the consequence of a variation of proinsulin 2 gene expression on the development of type 1 diabetes on the NOD genetic background. Heterozygote NOD mutant mice carrying a null proinsulin 2 mutation showed an increased incidence of type 1 diabetes at successive backcross generations. Plasma glucose and insulin levels were identical in prediabetic mutant and in wild-type mice at 4 weeks of age. Variation in insulin gene expression is hypothesized to interfere with diabetes development at both the islet and the thymus level.


Assuntos
Diabetes Mellitus Tipo 1/genética , Expressão Gênica , Variação Genética , Insulina/genética , Camundongos Endogâmicos NOD/fisiologia , Proinsulina/fisiologia , Animais , Predisposição Genética para Doença/genética , Camundongos , Camundongos Endogâmicos NOD/genética , Camundongos Knockout/genética , Proinsulina/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia
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