RESUMO
Endoplasmic reticulum (ER) stress is a key pathogenic factor in type 1 and 2 diabetes. Glycogen synthase kinase 3 (Gsk-3) contributes to ß-cell loss in mice. However, the mechanism by which Gsk-3 leads ß-cell death remains unclear. ER stress was pharmacologically induced in mouse primary islets and insulinoma cells. We used insulinoma cells derived from Akita mice as a model of genetic ER stress. Gsk-3 activity was blocked by treating with Gsk-3 inhibitors or by introducing catalytically inactive Gsk-3ß. Gsk-3 inhibition prevented proteasomal degradation of activating transcriptional factor 4 (ATF4) and alleviated apoptosis. We found that ATF4-S214 was phosphorylated by Gsk-3, and that this was required for a binding of ATF4 with ßTrCP, which mediates polyubiquitination. The anti-apoptotic effect of Gsk-3 inhibition was attenuated by introducing DN-ATF4 or by knockdown of ATF4. Mechanistically, Gsk-3 inhibition modulated transcription targets of ATF4 and in turn facilitated dephosphorylation of eIF2α, altering the protein translational dynamism under ER stress. These observations were reproduced in the Akita mouse-derived cells. Thus, these results reveal the role of Gsk-3 in the regulation of the integrated stress response, and provide a rationale for inhibiting this enzyme to prevent ß-cell death under ER stress conditions.
Assuntos
Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 2 , Insulinoma , Neoplasias Pancreáticas , Camundongos , Animais , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Transdução de Sinais , Estresse do Retículo Endoplasmático , ApoptoseRESUMO
Under conditions of high fat diet (HFD) consumption, glucose dyshomeostasis develops when ß-cells are unable to adapt to peripheral insulin demands. Few studies have interrogated the molecular mechanisms of ß-cell dysfunction at the level of mRNA translation under such conditions. We sought to address this issue through polyribosome profile analysis of islets from mice fed 16-weeks of 42% HFD. HFD-islet analysis revealed clear trends toward global reductions in mRNA translation with a significant reduction in the polyribosome/monoribosome ratio for Pdx1 mRNA. Transcriptional and translational analyses revealed endoplasmic reticulum stress was not the etiology of our findings. HFD-islets demonstrated evidence of oxidative stress and DNA damage, as well as activation of p53. Experiments in MIN-6 ß-cells revealed that treatment with doxorubicin to directly induce DNA damage mimicked our observed effects in islets. Islets from animals treated with pioglitazone concurrently with HFD demonstrated a reversal of effects observed from HFD alone. Finally, HFD-islets demonstrated reduced expression of multiple ribosome biogenesis genes and the key translation initiation factor eIF4E. We propose a heretofore unappreciated effect of chronic HFD on ß-cells, wherein continued DNA damage owing to persistent oxidative stress results in p53 activation and a resultant inhibition of mRNA translation.
Assuntos
Dano ao DNA , Gorduras na Dieta/efeitos adversos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , RNA Mensageiro/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Gorduras na Dieta/farmacologia , Doxorrubicina/farmacologia , Proteínas de Homeodomínio/biossíntese , Células Secretoras de Insulina/patologia , Masculino , Camundongos , Transativadores/biossínteseRESUMO
In type 1 diabetes, proinflammatory cytokines secreted by infiltrating immune cells activate the unfolded protein response (UPR) in islet ß-cells, which leads to attenuation of global mRNA translation. Under such conditions, privileged mRNAs required for adaptation to the prevailing stress are maintained in an actively translated state. Pdx1 is a ß-cell transcription factor that is required for the adaptive UPR, but it is not known how translation of its mRNA is maintained under these conditions. To study translation, we established conditions in vitro with MIN6 cells and mouse islets and a mixture of proinflammatory cytokines (IL-1ß, TNF-α, and IFN-γ) that mimicked the UPR conditions seen in type 1 diabetes. Cell extracts were then subjected to polyribosome profiling to monitor changes to mRNA occupancy by ribosomes. Similar to other privileged mRNAs (Atf4 and Chop), Pdx1 mRNA remained partitioned in actively translating polyribosomes under the UPR, whereas the mRNA encoding a proinsulin-processing enzyme (Cpe) and others partitioned into inactively translating monoribosomes. Bicistronic luciferase reporter analyses revealed that the distal portion of the 5'-untranslated region of mouse Pdx1 (between bp -105 to -280) contained elements that promoted translation under both normal and UPR conditions, and this region exhibited conserved sequences and secondary structure similar to those of other known internal ribosome entry sites. Our findings suggest that Pdx1 protein levels are maintained in the setting of the UPR, in part, through elements in the 5'-untranslated region that confer privileged mRNA translation in a 5'-7-methylguanylate cap-independent manner.
Assuntos
Proteínas de Homeodomínio/genética , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Transativadores/genética , Resposta a Proteínas não Dobradas/genética , Regiões 5' não Traduzidas/genética , Animais , Linhagem Celular Tumoral , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Interferon gama/metabolismo , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Polirribossomos/genética , Polirribossomos/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
An 80-year-old man presenting with abdominal distension was admitted to our hospital. He was diagnosed with sigmoid cancer with multiple liver and lung metastases. We first performed a sigmoidectomy to avoid obstruction, and then initiated chemotherapy with S-1(120mg/day). The tumor showed a complete clinical response after 10 courses, but we had to change the regimen after 18 courses because of growth of the lung metastases. After 10 courses of capecitabine(4,200mg/ day)treatment, we again observed growth of the lung metastases; a new nodule, which was also considered to be a metastasis, appeared on the abdominal wall. We then decided to administer mFOLFOX6(5-fluorouracil+Leucovorin+oxaliplatin) after the patient had received oral anticancer drugs for 3 years 4 month. In conclusion, oral chemotherapy drugs may prevent tumor growth over a long period and improve quality of life(QOL)in elderly patients with Stage IV colon cancer.
Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Desoxicitidina/análogos & derivados , Fluoruracila/análogos & derivados , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias do Colo Sigmoide/tratamento farmacológico , Administração Oral , Idoso , Capecitabina , Terapia Combinada , Desoxicitidina/uso terapêutico , Combinação de Medicamentos , Fluoruracila/uso terapêutico , Humanos , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/secundário , Masculino , Estadiamento de Neoplasias , Ácido Oxônico/administração & dosagem , Neoplasias do Colo Sigmoide/patologia , Neoplasias do Colo Sigmoide/cirurgia , Tegafur/administração & dosagem , Fatores de TempoRESUMO
Insulin secretion is precisely regulated by blood glucose with unique biphasic pattern. The regulatory mechanism of the second-phase insulin release is unclear. In this study, we report that DOC2b (double C2 domain protein isoform b), a SNARE related protein, was associated with insulin vesicles and translocated to plasma membrane within several minutes upon high-glucose stimulation followed by an interaction with syntaxin4, but not syntaxin1. This binding specificity and the time course of DOC2b translocation were suitable for the regulation of second-phase insulin release. Increased DOC2b expression enhanced glucose-stimulated insulin secretion. In contrast, silencing DOC2b inhibited delayed release of insulin, without affecting rapid (approximately 7min) phase secretion. Interestingly, DOC2b had no effects on KCl-triggered insulin release. These data suggest that DOC2b may be a regulator for delayed (second-phase) insulin secretion in MIN6 cells.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas SNARE/metabolismo , Animais , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Glucose/farmacologia , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/ultraestrutura , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Vesículas Secretórias/metabolismoRESUMO
To define the role of the unfolded protein response (UPR) in leukemogenesis, we investigated UPR activation in the cells expressing the representative oncogene Bcr-Abl (B-A). The expression of UPR-related proteins and mRNAs, namely, X-box-binding protein (XBP1) and glucose-regulated protein 78 (GRP78) was increased in B-A. UPR inhibition using inositol-requiring enzyme 1alpha (IRE1alpha) or activating transcription factor 6 (ATF6) dominant-negative mutants diminished the ability of Bcr-Abl to protect the cells from etoposide- and imatinib-induced apoptosis. We also noted that the expression of UPR-related genes in primary leukemia cells from Philadelphia chromosome (Ph)-positive cells was higher than that in the control by quantitative RT-PCR assay. Thus, our results suggested that UPR is a downstream target of Bcr-Abl and plays an anti-apoptotic role in Ph-positive leukemia cells.