RESUMO
Due to intrinsically low levels of antioxidant enzyme expression and activity, insulin producing pancreatic ß-cells are particularly susceptible to free radical attack. In diabetes mellitus, which is accompanied by high levels of oxidative stress, this feature of ß-cells significantly contributes to their damage and dysfunction. In light of the documented pro-survival effect of chemokine C-X-C Ligand 12 (CXCL12) on pancreatic ß-cells, we examined its potential role in antioxidant protection. We report that CXCL12 overexpression enhanced the resistance of rat insulinoma (Rin-5F) and primary pancreatic islet cells to hydrogen peroxide (H2O2). CXCL12 lowered the levels of DNA damage and lipid peroxidation and preserved insulin expression. This effect was mediated through an increase in catalase (CAT) activity. By activating downstream p38, Akt and ERK kinases, CXCL12 facilitated Nrf2 nuclear translocation and enhanced its binding to the CAT gene promoter, inducing constitutive CAT expression and activity that was essential for protecting ß-cells from H2O2.
Assuntos
Catalase/metabolismo , Quimiocina CXCL12/farmacologia , Citoproteção/efeitos dos fármacos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/enzimologia , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Animais , Antioxidantes/metabolismo , Sequência de Bases , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Peróxido de Hidrogênio/toxicidade , Células Secretoras de Insulina/efeitos dos fármacos , Masculino , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ligação Proteica , Ratos Wistar , Fatores de Transcrição/metabolismoRESUMO
Diabetes is characterized by a deficit in the number of functional pancreatic ß-cells. Understanding the mechanisms that stimulate neogenesis of ß-cells should contribute to improved maintenance of ß-cell mass. Chemokine CXCL12 has recently become established as a novel ß-cell growth factor, however the mechanisms controlling its expression require clarification. We investigated the proteins involved in the transcriptional regulation of the rat ß-cell CXCL12 gene (Cxcl12). Using the electrophoretic mobility shift assay and chromatin immunoprecipitation, we established the in vitro and in vivo binding of C/EBPß, C/EBPα, STAT3, p53, FOXO3a, and HMG I/Y to the Cxcl12 promoter. Co-immunoprecipitation experiments revealed protein-protein interactions between YY1 and PARP-1, FOXO3a and PARP-1, Sp1 and PARP-1, p53 and PARP-1, C/EBPß and PARP-1, YY1 and p53, YY1 and FOXO3a, p53 and FOXO3a, Sp1 and FOXO3a, C/EBPß and FOXO3a, C/EBPα and FOXO3a, Sp1 and STAT3. Our data lay the foundation for research into the interplay of signaling pathways that determine the ß-cell Cxcl12 expression profile.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Quimiocina CXCL12/genética , Células Secretoras de Insulina/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Sítios de Ligação , Proteína beta Intensificadora de Ligação a CCAAT/genética , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Ratos , Fator de Transcrição STAT3/genética , Ativação TranscricionalRESUMO
Introduction: Beta cell dysfunction by loss of beta cell identity, dedifferentiation, and the presence of polyhormonal cells are main characteristics of diabetes. The straightforward strategy for curing diabetes implies reestablishment of pancreatic beta cell function by beta cell replacement therapy. Aristaless-related homeobox (Arx) gene encodes protein which plays an important role in the development of pancreatic alpha cells and is a main target for changing alpha cell identity. Results: In this study we used CRISPR/dCas9-based epigenetic tools for targeted hypermethylation of Arx gene promoter and its subsequent suppression in mouse pancreatic αTC1-6 cell line. Bisulfite sequencing and methylation profiling revealed that the dCas9-Dnmt3a3L-KRAB single chain fusion constructs (EpiCRISPR) was the most efficient. Epigenetic silencing of Arx expression was accompanied by an increase in transcription of the insulin gene (Ins2) mRNA on 5th and 7th post-transfection day, quantified by both RT-qPCR and RNA-seq. Insulin production and secretion was determined by immunocytochemistry and ELISA assay, respectively. Eventually, we were able to induce switch of approximately 1% of transiently transfected cells which were able to produce 35% more insulin than Mock transfected alpha cells. Conclusion: In conclusion, we successfully triggered a direct, transient switch of pancreatic alpha to insulin-producing cells opening a future research on promising therapeutic avenue for diabetes management.
Assuntos
Diabetes Mellitus , Células Secretoras de Glucagon , Camundongos , Animais , Fatores de Transcrição/metabolismo , Proteínas de Homeodomínio/genética , Insulina/metabolismo , Células Secretoras de Glucagon/metabolismo , Metilação de DNA , Diabetes Mellitus/metabolismoRESUMO
BACKGROUND: Poly(ADP-ribosyl)ation (PARylation), a posttranslational modification introduced by PARP-1 and PARP-2, has first been implicated in DNA demethylation due to its role in base excision repair. Recent evidence indicates a direct influence of PARP-dependent PARylation on TET enzymes which catalyse hydroxymethylation of DNA-the first step in DNA demethylation. However, the exact nature of influence that PARylation exerts on TET activity is still ambiguous. In our recent study, we have observed a negative influence of PARP-1 on local TET-mediated DNA demethylation of a single gene and in this study, we further explore PARP-TET interplay. RESULTS: Expanding on our previous work, we show that both TET1 and TET2 can be in vitro PARylated by PARP-1 and PARP-2 enzymes and that TET1 PARylation negatively affects the TET1 catalytic activity in vitro. Furthermore, we show that PARylation inhibits TET-mediated DNA demethylation at the global genome level in cellulo. CONCLUSIONS: According to our findings, PARP inhibition can positively influence TET activity and therefore affect global levels of DNA methylation and hydroxymethylation. This gives a strong rationale for future examination of PARP inhibitors' potential use in the therapy of cancers characterised by loss of 5-hydroxymethylcytosine.
Assuntos
Poli ADP Ribosilação , Inibidores de Poli(ADP-Ribose) Polimerases , DNA/metabolismo , Metilação de DNA , Reparo do DNA , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologiaRESUMO
The present study aimed to investigate the beneficial effects of the treatment with extracts from the edible mushroom Lactarius deterrimus (Ld) and the chestnut Castanea sativa (Cs), separately and in combination (MIX Ld/Cs), on oxidative stress and advanced glycation end-product (AGE)-mediated hepatorenal injury in a rat model of streptozotocin (STZ)-induced diabetes by examining pathways responsible for maintenance of redox homeostasis. An experimental model of diabetes was induced in rats by the administration of 40 mg/kg STZ intraperitoneally (i.p.) for 5 consecutive days. The examined extracts were applied separately at a dose of 60 mg/kg i.p. and in combination (60 mg/kg each extract; i.p.) for 4 weeks, starting from the last day of STZ administration. The improvement of hepatorenal function in diabetic rats treated with the extracts was associated with an improved glycemic and lipid status and suppression of oxidative stress and thereby oxidative damage of lipids and DNA. Besides the fact that both extracts inhibited protein glycation and AGE formation in vitro, they also reduced non-enzymatic glycosylation in diabetic rats in vivo. The observed antiglycation activity of the examined extracts (separately and in combination) was accompanied with the inhibition of CML-mediated RAGE/NF-κB activation and reduction of enzymatic O-GlcNAcylation in liver and kidney tissues of diabetic rats. Taken together, these results reveal that the administration of chestnut and mushroom extracts, either individually or together, activates a coordinated cytoprotective response against diabetes-induced hepatorenal injury not only through recovery of the antioxidant defense system of the cell, but also through a marked antiglycation activity.
RESUMO
OBJECTIVES: CXCL12 is widely expressed, constitutive chemokine involved in tissue repair and regeneration, while the extent of its expression is important in various chronic inflammatory conditions. Involvement of DNA methylation in CXCL12 gene suppression (CXCL12) has been shown in malignancy and some autoimmune diseases. The aim of this study was to investigate whether the alterations in DNA methylation of CXCL12 are also involved in progression of periodontitis in combination with diabetes, as these chronic inflammatory conditions are strongly interrelated. DESIGN: Study included 72 subjects divided in three groups: healthy control (C, n=21), periodontitis (P, n=29) and diabetes/periodontitis group (D/P, n=22). DNA extracted from epithelial cells obtained by sterile cotton swabs from buccal mucosa was subjected to methylation specific polymerase chain reaction (MSP) to obtain DNA methylation pattern of CXCL12 promoter. RESULTS: CXCL12 promoter was predominantly unmethylated in all groups. However, increase in the frequency of the methylated form and increase in percent of methylation of CXCL12 promoter in periodontitis and diabetes/periodontitis group compared to control group were found, although without statistical significance. However, statistically significant increase in Tm of MSP products in diabetes/periodontitis group was observed. Correlation analysis revealed statistically significant relationship between the extent of DNA methylation of the CXCL12 promoter and periodontal parameters, as well as between DNA methylation of CXCL12 and glycosylated hemoglobin. CONCLUSION: Presented results suggest that chronic inflammation contributes to the change of CXCL12 DNA methylation in buccal cells and that DNA methylation profile of CXCL12 promoter plays important role in development and progression of periodontal disease.
Assuntos
Quimiocina CXCL12/genética , Periodontite Crônica/genética , Metilação de DNA/genética , Diabetes Mellitus Tipo 2/genética , Adulto , Estudos Transversais , Progressão da Doença , Feminino , Hemoglobinas Glicadas/genética , Humanos , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase em Tempo Real , Sérvia , Inquéritos e QuestionáriosRESUMO
The diabetes prevention paradigm envisages the application of strategies that support the maintenance of appropriate ß-cell numbers. Herein we show that overexpression of CXC chemokine ligand12 (CXCL12) considerably improves the viability of isolated rat Langerhans islet cells and Rin-5F pancreatic ß-cells after hydrogen peroxide treatment. In rat islets and wt cells hydrogen peroxide treatment induced necrotic cell death that was mediated by the rapid and extensive activation of poly(ADP-ribose) polymerase-1 (PARP-1). In contrast, CXCL12-overexpressing cells were protected from necrotic cell death as a result of significantly reduced PARP-1 activity. CXCL12 downstream signalling through Akt kinase was responsible for the reduction of PARP-1 activity which switched cell death from necrosis to apoptosis, providing increased protection to cells from oxidative stress. Our results offer a novel aspect of the CXCL12-mediated improvement of ß-cell viability which is based on its antinecrotic action through modulation of PARP-1 activity.