RESUMO
BACKGROUND: Type I Diabetes mellitus (T1D) is characterized by a specific destruction of ß-cells by the immune system. During this process pro-inflammatory cytokines are released in the pancreatic islets and contribute for ß-cells demise. Cytokine-induced iNOS activation, via NF-κB, is implicated in induction of ß-cells death, which includes ER stress activation. Physical exercise has been used as an adjunct for better glycemic control in patients with T1D, since it is able to increase glucose uptake independent of insulin. Recently, it was observed that the release of IL-6 by skeletal muscle, during physical exercise, could prevent ß-cells death induced by pro-inflammatory cytokines. However, the molecular mechanisms involved in this beneficial effect on ß-cells are not yet completely elucidated. Our aim was to evaluate the effect of IL-6 on ß-cells exposed to pro-inflammatory cytokines. RESULTS: Pre-treatment with IL-6 sensitized INS-1E cells to cytokine-induced cell death, increasing cytokine-induced iNOS and Caspase-3 expression. Under these conditions, however, there was a decrease in cytokines-induced p-eIF2-α but not p-IRE1expression, proteins related to ER stress. To address if this prevention of adequate UPR response is involved in the increase in ß-cells death markers induced by IL-6 pre-treatment, we used a chemical chaperone (TUDCA), which improves ER folding capacity. Use of TUDCA increased cytokines-induced Caspase-3 expression and Bax/Bcl-2 ratio in the presence of IL-6 pre-treatment. However, there is no modulation of p-eIF2-α expression by TUDCA in this condition, with increase of CHOP expression. CONCLUSION: Treatment with IL-6 alone is not beneficial for ß-cells, leading to increased cell death markers and impaired UPR activation. In addition, TUDCA has not been able to restore ER homeostasis or improve ß-cells viability under this condition, suggesting that other mechanisms may be involved.
Assuntos
Citocinas , Diabetes Mellitus Tipo 1 , Humanos , Citocinas/metabolismo , Caspase 3 , Interleucina-6/farmacologia , Diabetes Mellitus Tipo 1/metabolismo , Fator de Iniciação 2 em Eucariotos/farmacologia , Morte Celular/fisiologiaRESUMO
Diets rich in saturated fats may contribute to the loss of pancreatic ß-cells in type 2 diabetes. JunB, a member of the activating protein 1 (AP-1) transcription factor family, promotes ß-cell survival and mediates part of the beneficial effects of GLP-1 agonists. In this study we interrogated the molecular mechanisms involved in JunB-mediated ß-cell protection from lipotoxicity. The saturated fatty acid palmitate decreased JunB expression, and this loss may contribute to ß-cell apoptosis, as overexpression of JunB protected cells from lipotoxicity. Array analysis of JunB-deficient ß-cells identified a gene expression signature of a downregulated endoplasmic reticulum (ER) stress response and inhibited AKT signaling. JunB stimulates XBP1 expression via the transcription factor c/EBPδ during ER stress, and forced expression of XBP1s rescued the viability of JunB-deficient cells, constituting an important antiapoptotic mechanism. JunB silencing inhibited AKT activation and activated the proapoptotic Bcl-2 protein BAD via its dephosphorylation. BAD knockdown reversed lipotoxic ß-cell death potentiated by JunB siRNA. Interestingly, XBP1s links JunB and AKT signaling as XBP1 knockdown also reduced AKT phosphorylation. GLP-1 agonists induced cAMP-dependent AKT phosphorylation leading to ß-cell protection against palmitate-induced apoptosis. JunB and XBP1 knockdown or IRE1 inhibition decreased AKT activation by cAMP, leading to ß-cell apoptosis. In conclusion, JunB modulates the ß-cell ER stress response and AKT signaling via the induction of XBP1s. The activation of the JunB gene network and the crosstalk between the ER stress and AKT pathway constitute a crucial defense mechanism by which GLP-1 agonists protect against lipotoxic ß-cell death. These findings elucidate novel ß-cell-protective signal transduction in type 2 diabetes.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Células Secretoras de Insulina/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Humanos , Células Secretoras de Insulina/enzimologia , Masculino , Pessoa de Meia-Idade , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Fatores de Transcrição de Fator Regulador X , Transdução de Sinais , Fatores de Transcrição/genética , Proteína 1 de Ligação a X-BoxRESUMO
The cytokines interleukin (IL)-1ß and tumor necrosis factor (TNF)-α induce ß-cell death in type 1 diabetes via NF-κB activation. IL-1ß induces a more marked NF-κB activation than TNF-α, with higher expression of genes involved in ß-cell dysfunction and death. We show here a differential usage of the IKK complex by IL-1ß and TNF-α in ß-cells. While TNF-α uses IKK complexes containing both IKKα and IKKß, IL-1ß induces complexes with IKKα only; this effect is achieved by induction of IKKß degradation via the proteasome. Both IKKγ and activation of the TRAF6-TAK1-JNK pathway are involved in IL-1ß-induced IKKß degradation.
Assuntos
Quinase I-kappa B/metabolismo , Células Secretoras de Insulina/metabolismo , Interleucina-1beta/metabolismo , NF-kappa B/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Diabetes Mellitus Tipo 1/tratamento farmacológico , Inativação Gênica , Humanos , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/genética , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos , Terapia de Alvo Molecular , Inibidores de Proteases/farmacologia , Inibidores de Proteassoma , Inibidores de Proteínas Quinases/farmacologia , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteólise/efeitos dos fármacos , Ratos , Ratos Wistar , Proteínas Recombinantes/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genéticaRESUMO
Destruction of insulin-producing pancreatic ß-cells by local autoimmune inflammation is a hallmark of type 1 diabetes. Histochemical analysis of pancreases from non-obese diabetic mice indicated activation of the transcription factor JunB/AP-1 (activator protein-1) after autoimmune infiltration of the islets. In vitro studies demonstrated that the cytokines tumor necrosis factor (TNF)-α and interferon (IFN)-γ induce JunB expression as a protective mechanism against apoptosis in both human and rodent ß-cells. The gene network affected was studied by microarray analysis showing that JunB regulates nearly 20% of the cytokine-modified ß-cell genes, including the transcription factor ATF3. Direct transcriptional induction of ATF3 by JunB is a key event for ß-cell survival after TNF-α+IFN-γ treatment. Moreover, pharmacological upregulation of JunB/ATF3 via increased cAMP protected rodent primary ß-cells and human islet cells against pro-inflammatory mediators. These results were confirmed in genetically modified islets derived from Ubi-JunB transgenic mice. Our findings identify ATF3 as a novel downstream target of JunB in the survival mechanism of ß-cells under inflammatory stress.
Assuntos
Fator 3 Ativador da Transcrição/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Inflamação/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Animais , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-jun/genética , Transdução de Sinais , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
AIMS/HYPOTHESIS: Beta cell failure is a crucial component in the pathogenesis of type 2 diabetes. One of the proposed mechanisms of beta cell failure is local inflammation, but the presence of pancreatic islet inflammation in type 2 diabetes and the mechanisms involved remain under debate. METHODS: Chemokine and cytokine expression was studied by microarray analysis of laser-capture microdissected islets from pancreases obtained from ten non-diabetic and ten type 2 diabetic donors, and by real-time PCR of human islets exposed to oleate or palmitate at 6 or 28 mmol/l glucose. The cellular source of the chemokines was analysed by immunofluorescence of pancreatic sections from individuals without diabetes and with type 2 diabetes. RESULTS: Microarray analysis of laser-capture microdissected beta cells showed increased chemokine and cytokine expression in type 2 diabetes compared with non-diabetic controls. The inflammatory response in type 2 diabetes was mimicked by exposure of non-diabetic human islets to palmitate, but not to oleate or high glucose, leading to the induction of IL-1beta, TNF-alpha, IL-6, IL-8, chemokine (C-X-C motif) ligand 1 (CXCL1) and chemokine (C-C motif) ligand 2 (CCL2). Interference with IL-1beta signalling abolished palmitate-induced cytokine and chemokine expression but failed to prevent lipotoxic human islet cell death. Palmitate activated nuclear factor kappaB (NF-kappaB) in human pancreatic beta and non-beta cells, and chemically induced endoplasmic reticulum stress caused cytokine expression and NF-kappaB activation similar to that occurring with palmitate. CONCLUSIONS/INTERPRETATION: Saturated-fatty-acid-induced NF-kappaB activation and endoplasmic reticulum stress may contribute to IL-1beta production and mild islet inflammation in type 2 diabetes. This inflammatory process does not contribute to lipotoxicity ex vivo, but may lead to local chemokine release.
Assuntos
Quimiocina CCL2/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Palmitatos/farmacologia , Idoso , Linhagem Celular , Quimiocina CXCL1 , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Humanos , Técnicas In Vitro , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Masculino , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Radioimunoensaio , Fator de Necrose Tumoral alfa/metabolismoRESUMO
AIMS/HYPOTHESIS: Pro-inflammatory cytokines involved in the pathogenesis of type 1 diabetes deplete endoplasmic reticulum (ER) Ca2+ stores, leading to ER-stress and beta cell apoptosis. However, the cytokine-induced ER-stress response in beta cells is atypical and characterised by induction of the pro-apoptotic PKR-like ER kinase (PERK)-C/EBP homologous protein (CHOP) branch of the unfolded protein response, but defective X-box binding protein 1 (XBP1) splicing and activating transcription factor 6 activation. The purpose of this study was to overexpress spliced/active Xbp1 (XBP1s) to increase beta cell resistance to cytokine-induced ER-stress and apoptosis. METHODS: Xbp1s was overexpressed using adenoviruses and knocked down using small interference RNA in rat islet cells. In selected experiments, Xbp1 was also knocked down in FACS-purified rat beta cells and rat fibroblasts. Expression and production of XBP1s and key downstream genes and proteins was measured and beta cell function and viability were evaluated. RESULTS: Adenoviral-mediated overproduction of Xbp1s resulted in increased XBP1 activity and induction of several XBP1s target genes. Surprisingly, XBP1s overexpression impaired glucose-stimulated insulin secretion and increased beta cell apoptosis, whereas it protected fibroblasts against cell death induced by ER-stress. mRNA expression of Pdx1 and Mafa was inhibited in cells overproducing XBP1s, leading to decreased insulin expression. XBP1s knockdown partially restored cytokine/ER-stress-driven insulin and Pdx1 inhibition but had no effect on cytokine-induced ER-stress and apoptosis. CONCLUSIONS/INTERPRETATION: XBP1 has a distinct inhibitory role in beta cell as compared with other cell types. Prolonged XBP1s production hampers beta cell function via inhibition of insulin, Pdx1 and Mafa expression, eventually leading to beta cell apoptosis.
Assuntos
Apoptose/fisiologia , Proteínas de Ligação a DNA/metabolismo , Retículo Endoplasmático/metabolismo , Células Secretoras de Insulina/metabolismo , Fatores de Transcrição/metabolismo , Análise de Variância , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Contagem de Células , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Proteínas de Ligação a DNA/genética , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/genética , Imunofluorescência , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Indóis/farmacologia , Insulina/genética , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Interferon gama/farmacologia , Interleucina-8/farmacologia , Fatores de Transcrição Maf/genética , Fatores de Transcrição Maf/metabolismo , Masculino , Interferência de RNA , RNA Interferente Pequeno , Ratos , Ratos Wistar , Fatores de Transcrição de Fator Regulador X , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Transfecção , Proteína 1 de Ligação a X-BoxRESUMO
Chronic inflammation and pro-inflammatory cytokines are important mediators of pancreatic beta-cell destruction in type 1 diabetes (T1D). We presently show that the cytokines IL-1beta+IFN-gamma and different ER stressors activate the Bcl-2 homology 3 (BH3)-only member death protein 5 (DP5)/harakiri (Hrk) resulting in beta-cell apoptosis. Chemical ER stress-induced DP5 upregulation is JNK/c-Jun-dependent. DP5 activation by cytokines also involves JNK/c-Jun phosphorylation and is antagonized by JunB. Interestingly, cytokine-inducted DP5 expression precedes ER stress: mitochondrial release of cytochrome c and ER stress are actually a consequence of enhanced DP5 activation by cytokine-mediated nitric oxide formation. Our findings show that DP5 is central for beta-cell apoptosis after different stimuli, and that it can act up- and downstream of ER stress. These observations contribute to solve two important questions, namely the mechanism by which IL-1beta+IFN-gamma induce beta-cell death and the nature of the downstream signals by which ER stress 'convinces' beta-cells to trigger apoptosis.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Retículo Endoplasmático/metabolismo , Células Secretoras de Insulina/citologia , Interferon gama/farmacologia , Interleucina-1beta/farmacologia , Neuropeptídeos/metabolismo , Animais , Citocromos c/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais , Regulação para CimaRESUMO
AIMS/HYPOTHESIS: IL-1beta and TNF-alpha contribute to pancreatic beta cell death in type 1 diabetes. Both cytokines activate the transcription factor nuclear factor-kappaB (NF-kappaB), but recent observations suggest that NF-kappaB blockade prevents IL-1beta + IFN-gamma- but not TNF-alpha + IFN-gamma-induced beta cell apoptosis. The aim of the present study was to compare the effects of IL-1beta and TNF-alpha on cell death and the pattern of NF-kappaB activation and global gene expression in beta cells. METHODS: Cell viability was measured after exposure to IL-1beta or to TNF-alpha alone or in combination with IFN-gamma, and blockade of NF-kappaB activation or protein synthesis. INS-1E cells exposed to IL-1beta or TNF-alpha in time course experiments were used for IkappaB kinase (IKK) activation assay, detection of p65 NF-kappaB by immunocytochemistry, real-time RT-PCR and microarray analysis. RESULTS: Blocking NF-kappaB activation protected beta cells against IL-1beta + IFNgamma- or TNFalpha + IFNgamma-induced apoptosis. Blocking de novo protein synthesis did not increase TNF-alpha- or IL-1beta-induced beta cell death, in line with the observations that cytokines induced the expression of the anti-apoptotic genes A20, Iap-2 and Xiap to a similar extent. Microarray analysis of INS-1E cells treated with IL-1beta or TNF-alpha showed similar patterns of gene expression. IL-1beta, however, induced a higher rate of expression of NF-kappaB target genes putatively involved in beta cell dysfunction and death and a stronger activation of the IKK complex, leading to an earlier translocation of NF-kappaB to the nucleus. CONCLUSIONS/INTERPRETATION: NF-kappaB activation in beta cells has a pro-apoptotic role following exposure not only to IL-1beta but also to TNF-alpha. The more marked beta cell death induced by IL-1beta is explained at least in part by higher intensity NF-kappaB activation, leading to increased transcription of key target genes.
Assuntos
Apoptose/fisiologia , Diabetes Mellitus Tipo 1/fisiopatologia , Células Secretoras de Insulina/fisiologia , Interleucina-1beta/metabolismo , NF-kappa B/genética , Fator de Necrose Tumoral alfa/metabolismo , Animais , Apoptose/efeitos dos fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Quinase I-kappa B/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Interleucina-1beta/farmacologia , NF-kappa B/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
In order to study the role played by known and novel genes in growth control and neoplasia, we here compare the pEX and pGEX bacterial expression systems for recombinant oncoprotein production and for generation of specific antisera. The results of five pEX (MS2-c-Fos, MS2-Fra-1, MS2-JunD, bgal-c-Jun and bgal-JunB) and two pGEX [glutathione S-transferase (GSH)-JE/MCP-1 and GST-JunD] fusion-protein productions are presented. Higher (15-43-fold) yields are obtained with the pEX system, but only the pGEX system allows separation of the protein of interest from the fusion moiety by digestion with specific proteases. The degree of fusion-protein purification, as assessed by SDS/PAGE, is similar for both systems. Proteins produced by both systems were successfully used in the generation of specific antisera. The choice between the pEX and pGEX systems is dependent upon the specific recombinant protein produced.