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AIMS/HYPOTHESIS: The proinflammatory cytokines IFN-α, IFN-γ, IL-1ß and TNF-α may contribute to innate and adaptive immune responses during insulitis in type 1 diabetes and therefore represent attractive therapeutic targets to protect beta cells. However, the specific role of each of these cytokines individually on pancreatic beta cells remains unknown. METHODS: We used deep RNA-seq analysis, followed by extensive confirmation experiments based on reverse transcription-quantitative PCR (RT-qPCR), western blot, histology and use of siRNAs, to characterise the response of human pancreatic beta cells to each cytokine individually and compared the signatures obtained with those present in islets of individuals affected by type 1 diabetes. RESULTS: IFN-α and IFN-γ had a greater impact on the beta cell transcriptome when compared with IL-1ß and TNF-α. The IFN-induced gene signatures have a strong correlation with those observed in beta cells from individuals with type 1 diabetes, and the level of expression of specific IFN-stimulated genes is positively correlated with proteins present in islets of these individuals, regulating beta cell responses to 'danger signals' such as viral infections. Zinc finger NFX1-type containing 1 (ZNFX1), a double-stranded RNA sensor, was identified as highly induced by IFNs and shown to play a key role in the antiviral response in beta cells. CONCLUSIONS/INTERPRETATION: These data suggest that IFN-α and IFN-γ are key cytokines at the islet level in human type 1 diabetes, contributing to the triggering and amplification of autoimmunity.
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Diabetes Mellitus Tipo 1 , Ilhotas Pancreáticas , Humanos , Citocinas/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Interferons/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Interferon gama/metabolismo , Ilhotas Pancreáticas/metabolismoRESUMO
AIMS/HYPOTHESIS: Diabetes is characterised by progressive loss of functional pancreatic beta cells. None of the therapeutic agents used to treat diabetes arrest this process; preventing beta cell loss remains a major unmet need. We have previously shown that serum from eight young healthy male participants who exercised for 8 weeks protected human islets and insulin-producing EndoC-ßH1 cells from apoptosis induced by proinflammatory cytokines or the endoplasmic reticulum (ER) stressor thapsigargin. Whether this protective effect is influenced by sex, age, training modality, ancestry or diabetes is unknown. METHODS: We enrolled 82 individuals, male or female, non-diabetic or diabetic, from different origins, in different supervised training protocols for 8-12 weeks (including training at home during the COVID-19 pandemic). EndoC-ßH1 cells were treated with 'exercised' serum or with the exerkine clusterin to ascertain cytoprotection from ER stress. RESULTS: The exercise interventions were effective and improved [Formula: see text] values in both younger and older, non-obese and obese, non-diabetic and diabetic participants. Serum obtained after training conferred significant beta cell protection (28% to 35% protection after 4 and 8 weeks of training, respectively) from severe ER stress-induced apoptosis. Cytoprotection was not affected by the type of exercise training or participant age, sex, BMI or ancestry, and persisted for up to 2 months after the end of the training programme. Serum from exercised participants with type 1 or type 2 diabetes was similarly protective. Clusterin reproduced the beneficial effects of exercised sera. CONCLUSIONS/INTERPRETATION: These data uncover the unexpected potential to preserve beta cell health by exercise training, opening a new avenue to prevent or slow diabetes progression through humoral muscle-beta cell crosstalk.
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COVID-19 , Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Humanos , Masculino , Feminino , Lactente , Células Secretoras de Insulina/metabolismo , Diabetes Mellitus Tipo 2/terapia , Diabetes Mellitus Tipo 2/metabolismo , Clusterina/metabolismo , Clusterina/farmacologia , Pandemias , Apoptose/fisiologia , Estresse do Retículo EndoplasmáticoRESUMO
AIM: Type 1 diabetes (T1D) is a chronic autoimmune disease leading to progressive loss of pancreatic beta cells. Interferon (IFN)-α plays a critical role in the crosstalk between pancreatic beta cells and the immune system in early insulitis. In human beta cells IFNα signals through JAK1 and TYK2, leading to endoplasmic reticulum stress, inflammation and HLA class I overexpression. IFNα, acting synergistically with IL-1ß, induces apoptosis. Polymorphisms in TYK2 that decrease its activity are associated with protection against T1D, and we hypothesized that pharmacological inhibitors that specifically target TYK2 could protect human beta cells against the deleterious effects of IFNα. MATERIALS AND METHODS: Two TYK2 inhibitors provided by Nimbus Lakshmi were tested in human insulin-producing EndoC-ßH1 cells and human islets to evaluate their effect on IFNα signalling, beta-cell function and susceptibility to viral infection using RT-qPCR, western blot, immunofluorescence, ELISA and nuclear dyes. RESULTS: The two TYK2 inhibitors tested prevented IFNα-induced human beta-cell gene expression in a dose-dependent manner. They also protected human islets against IFNα + IL-1ß-induced apoptosis. Importantly, these inhibitors did not modify beta-cell function or their survival following infection with the potential diabetogenic coxsackieviruses CVB1 and CVB5. CONCLUSIONS: The two TYK2 inhibitors tested inhibit the IFNα signalling pathway in human beta cells, decreasing its pro-inflammatory and pro-apoptotic effects without sensitizing the cells to viral infection. The preclinical findings could pave the way for future clinical trials with TYK2 inhibitors for the prevention and treatment of type 1 diabetes.
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Diabetes Mellitus Tipo 1 , Células Secretoras de Insulina , Apoptose , Citoproteção , Diabetes Mellitus Tipo 1/tratamento farmacológico , Estresse do Retículo Endoplasmático , Humanos , TYK2 Quinase/genéticaRESUMO
Erectile dysfunction (ED) is a complication of diabetes, condition responsible for causing endothelial dysfunction (EDys) and hampering repair mechanisms. However, scarce information is available linking vasculogenesis mediated by Endothelial Progenitor Cells (EPCs) and diabetes-associated ED. Furthermore, it remains to be elucidated if glycemic control plays a role on EPCs functions, EPCs modulators, and penile vascular health. We evaluated the effects of diabetes and insulin therapy on bone marrow (BM) and circulating EPCs, testosterone, and systemic/penile Stromal Derived Factor-1 alpha (SDF-1α) expression. Male Wistar rats were divided into groups: age-matched controls, 8-weeks streptozotocin-induced type 1 diabetics, and insulin-treated 8-weeks diabetics. EPCs were identified by flow cytometry for CD34/CD133/VEGFR2/CXCR4 antigens. Systemic SDF-1α and testosterone levels were evaluated by ELISA. Penile SDF-1α protein expression was assessed, in experimental and human diabetic cavernosal samples, by immunohistochemical techniques. Diabetic animals presented a reduction of BM-derived EPCs and an increase in putative circulating endothelial cells (CECs) sloughed from vessels wall. These alterations were rescued by insulin therapy. In addition, glycemic control promoted an increase in systemic testosterone and SDF-1α levels, which were significantly decreased in animals with diabetes. SDF-1α protein expression was reduced in experimental and human cavernosal diabetic samples, an effect prevented by insulin in treated animals. Insulin administration rescued the effects of diabetes on BM function, CECs levels, testosterone, and plasmatic/penile SDF-1α protein expression. This emphasizes the importance of glycemic control in the prevention of diabetes-induced systemic and penile EDys, by the amelioration of endothelial damage, and increase in protective pathways. J. Cell. Biochem. 118: 82-91, 2017. © 2016 Wiley Periodicals, Inc.
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Quimiocina CXCL12/sangue , Complicações do Diabetes/sangue , Diabetes Mellitus Experimental/sangue , Impotência Vasculogênica/sangue , Testosterona/sangue , Animais , Complicações do Diabetes/terapia , Diabetes Mellitus Experimental/terapia , Impotência Vasculogênica/prevenção & controle , Masculino , Ratos , Ratos WistarRESUMO
INTRODUCTION: Novel effective therapeutic strategies are necessary for treating erectile dysfunction secondary to cavernous nerve injury (CNI). AIM: To functionally evaluate the benefits of long-term oral treatment with a phosphodiesterase type 5 inhibitor on the potential capacity of intracavernosal cell therapy to recover erectile function after CNI. METHODS: Bilateral crush CNI (BCNI) was produced in anesthetized male rats. After BCNI, rats were treated with the phosphodiesterase type 5 inhibitor tadalafil (TAD; 5 mg/kg/d orally; BCNI + TAD), a single intracavernosal injection of bone marrow-derived mesenchymal stem cells (BMSCs; BCNI + BMSC), or dual therapy (BCNI + BMSC + TAD). Ex vivo function of the corpus cavernosum (CC) and in vivo intracavernosal pressure responses to CN electrical stimulation were evaluated 4 weeks after BCNI. Trichrome staining and terminal 2'-deoxyuridine-5'-triphosphate nick-end labeling assay were used for fibrosis and apoptosis determination, respectively, in the CC. MAIN OUTCOME MEASURES: In vivo erectile responses in anesthetized rats, ex vivo evaluation of endothelium-dependent relaxation, neurogenic relaxation and neurogenic contraction in CC strips, and histologic evaluation of fibrosis and apoptosis in cavernosal tissue. RESULTS: BCNI resulted in a marked decrease of erectile responses that were partly recovered in the BCNI + TAD and BCNI + BMSC groups. Complete recovery of erectile function was achieved only in the BCNI + BMSC + TAD group. Endothelium-dependent and nitric oxide donor-induced relaxations of the CC were not altered by BCNI or the treatments. BCNI resulted in enhanced neurogenic adrenergic contractions and impaired nitrergic relaxations of the CC. The BCNI + TAD group displayed diminished neurogenic contractions, whereas the BCNI + TAD and BCNI + BMSC groups showed partly recovered nitrergic responses. In the BCNI + BMSC + TAD group, neurogenic contractions were decreased and nitrergic relaxations were normalized. Cavernosal apoptosis and fibrosis were similarly prevented in the BCNI + TAD, BCNI + BMSC, and BCNI + BMSC + TAD groups. CONCLUSION: A dual strategy combining the intracavernosal injection of BMSCs and oral administration of TAD was superior to individual approaches in normalizing neurogenic control of cavernosal tone and preserving erectile function after CNI, suggesting the potential of this dual strategy in the future management of erectile dysfunction after radical prostatectomy.
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Disfunção Erétil/patologia , Células-Tronco Mesenquimais/metabolismo , Ereção Peniana/efeitos dos fármacos , Pênis/patologia , Traumatismos dos Nervos Periféricos/patologia , Inibidores da Fosfodiesterase 5/farmacologia , Tadalafila/farmacologia , Animais , Terapia Combinada , Modelos Animais de Doenças , Disfunção Erétil/tratamento farmacológico , Masculino , Compressão Nervosa , Prostatectomia/efeitos adversos , RatosRESUMO
Tyrosine protein-kinase 2 (TYK2), a member of the Janus kinase family, mediates inflammatory signaling through multiple cytokines, including interferon-α (IFNα), interleukin (IL)-12, and IL-23. Missense mutations in TYK2 are associated with protection against type 1 diabetes (T1D), and inhibition of TYK2 shows promise in the management of other autoimmune conditions. Here, we evaluated the effects of specific TYK2 inhibitors (TYK2is) in pre-clinical models of T1D. First, human ß cells, cadaveric donor islets, and iPSC-derived islets were treated in vitro with IFNα in combination with a small molecule TYK2i (BMS-986165 or a related molecule BMS-986202). TYK2 inhibition prevented IFNα-induced ß cell HLA class I up-regulation, endoplasmic reticulum stress, and chemokine production. In co-culture studies, pre-treatment of ß cells with a TYK2i prevented IFNα-induced activation of T cells targeting an epitope of insulin. In vivo administration of BMS-986202 in two mouse models of T1D (RIP-LCMV-GP mice and NOD mice) reduced systemic and tissue-localized inflammation, prevented ß cell death, and delayed T1D onset. Transcriptional phenotyping of pancreatic islets, pancreatic lymph nodes (PLN), and spleen during early disease pathogenesis highlighted a role for TYK2 inhibition in modulating signaling pathways associated with inflammation, translational control, stress signaling, secretory function, immunity, and diabetes. Additionally, TYK2i treatment changed the composition of innate and adaptive immune cell populations in the blood and disease target tissues, resulting in an immune phenotype with a diminished capacity for ß cell destruction. Overall, these findings indicate that TYK2i has beneficial effects in both the immune and endocrine compartments in models of T1D, thus supporting a path forward for testing TYK2 inhibitors in human T1D.
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Type 1 diabetes mellitus is responsible for metabolic dysfunction, accompanied by chronic inflammation, oxidative stress, and endothelium dysfunction, and is often associated with impaired wound healing. Phenol-rich food improves vascular function, contributing to diabetes prevention. This study has evaluated the effect of phenol-rich beverage consumption in diabetic rats on wound healing, through angiogenesis, inflammation, and oxidative stress modulation. A wound-healing assay was performed in streptozotocin-induced diabetic Wistar rats drinking water, 5% ethanol, and stout beer with and without 10 mg/L xanthohumol (1), for a five-week period. Wounded skin microvessel density was reduced to normal values upon consumption of 1 in diabetic rats, being accompanied by decreased serum VEGF-A and inflammatory markers (IL-1ß, NO, N-acetylglucosaminidase). Systemic glutathione and kidney and liver H2O2, 3-nitrotyrosine, and protein carbonylation also decreased to healthy levels after treatment with 1, implying an improvement in oxidative stress status. These findings suggest that consumption of xanthohumol (1) by diabetic animals consistently decreases inflammation and oxidative stress, allowing neovascularization control and improving diabetic wound healing.
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Indutores da Angiogênese/metabolismo , Flavonoides/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Propiofenonas/farmacologia , Animais , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Flavonoides/química , Glutationa/metabolismo , Peróxido de Hidrogênio/metabolismo , Inflamação/complicações , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Fígado/metabolismo , Masculino , Neovascularização Patológica/metabolismo , Fenóis/farmacologia , Propiofenonas/química , Ratos , Ratos Wistar , Pele/efeitos dos fármacos , Tirosina/análogos & derivados , Fator A de Crescimento do Endotélio Vascular/metabolismo , Cicatrização/efeitos dos fármacosRESUMO
Diabetes-induced metabolic derangements promote endothelial malfunction, contributing to erectile dysfunction (ED). However, it remains unclear which angiogenic molecular mechanisms are deregulated in diabetic corpus cavernosum (CC). We investigated early and late alterations in cavernosal angiogenic gene expression associated to diabetes. Angiogenic changes were assessed in penile tissue of streptozotocin-induced Wistar rats, in an early (2-week) and established stage (8-week) of diabetes. Differentially expressed genes were identified by microarrays and expression data validated by quantitative real-time PCR (qrt-PCR). At protein level, quantitative immunohistochemistry confirmed the arrays data and dual immunofluorescence for selected alterations and α-smooth muscle actin (α-SMA) identified the cellular location of target proteins. The selected differentially expressed genes were also evaluated in human non-diabetic and diabetic CC by quantitative immunolabeling. At 2-week diabetes there was no differential gene expression between non-diabetic and diabetic CC. At 8-week, 10 genes were found down-regulated in diabetics. The results were validated by qrt-PCR for the insulin-like growth factor-1 (Igf1) and the natriuretic peptide receptor-1 (Npr1) genes. Dual immunofluorescence for IGF-1/ α-SMA showed predominant localization of IGF-1 in SM. NPR-1 expression was diffuse and mostly present in trabecular fibroblasts and SM. Quantitative immunostaining confirmed the decreased expression of both proteins in diabetic tissues. Concordantly, we detected a significant reduction in IGF-1 and NPR-1 protein expressions in human diabetic samples. Microarray analysis identified 10 angiogenic-related molecules deregulated in CC of established diabetes. Among them, IGF-1 and NPR-1 were significantly down-regulated and might result in preventive/therapeutic targets for ED management.
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Actinas/genética , Actinas/metabolismo , Corpo Caloso/patologia , Complicações do Diabetes/fisiopatologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus/metabolismo , Disfunção Erétil/fisiopatologia , Fator de Crescimento Insulin-Like I/metabolismo , Receptores do Fator Natriurético Atrial/metabolismo , Animais , Corpo Caloso/metabolismo , Complicações do Diabetes/genética , Complicações do Diabetes/metabolismo , Diabetes Mellitus/genética , Diabetes Mellitus Experimental/genética , Endotélio/metabolismo , Endotélio/patologia , Disfunção Erétil/genética , Disfunção Erétil/metabolismo , Expressão Gênica , Humanos , Fator de Crescimento Insulin-Like I/genética , Masculino , Pessoa de Meia-Idade , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Ratos Wistar , Receptores do Fator Natriurético Atrial/genéticaRESUMO
Introduction: Enterovirus infection has long been suspected as a possible trigger for type 1 diabetes. Upon infection, viral double-stranded RNA (dsRNA) is recognized by membrane and cytosolic sensors that orchestrate type I interferon signaling and the recruitment of innate immune cells to the pancreatic islets. In this context, adenosine deaminase acting on RNA 1 (ADAR1) editing plays an important role in dampening the immune response by inducing adenosine mispairing, destabilizing the RNA duplexes and thus preventing excessive immune activation. Methods: Using high-throughput RNA sequencing data from human islets and EndoC-ßH1 cells exposed to IFNα or IFNγ/IL1ß, we evaluated the role of ADAR1 in human pancreatic ß cells and determined the impact of the type 1 diabetes pathophysiological environment on ADAR1-dependent RNA editing. Results: We show that both IFNα and IFNγ/IL1ß stimulation promote ADAR1 expression and increase the A-to-I RNA editing of Alu-Containing mRNAs in EndoC-ßH1 cells as well as in primary human islets. Discussion: We demonstrate that ADAR1 overexpression inhibits type I interferon response signaling, while ADAR1 silencing potentiates IFNα effects. In addition, ADAR1 overexpression triggers the generation of alternatively spliced mRNAs, highlighting a novel role for ADAR1 as a regulator of the ß cell transcriptome under inflammatory conditions.
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Adenosina Desaminase , Diabetes Mellitus Tipo 1 , Células Secretoras de Insulina , Interferon Tipo I , Proteínas de Ligação a RNA , Humanos , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Inflamação/genética , Células Secretoras de Insulina/metabolismo , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , RNA de Cadeia Dupla , RNA Mensageiro , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , TranscriptomaRESUMO
IFNα is a key regulator of the dialogue between pancreatic ß cells and the immune system in early type 1 diabetes (T1D). IFNα up-regulates HLA class I expression in human ß cells, fostering autoantigen presentation to the immune system. We observed by bulk and single-cell RNA sequencing that exposure of human induced pluripotent-derived islet-like cells to IFNα induces expression of HLA class I and of other genes involved in antigen presentation, including the transcriptional activator NLRC5. We next evaluated the global role of NLRC5 in human insulin-producing EndoC-ßH1 and human islet cells by RNA sequencing and targeted gene/protein determination. NLRC5 regulates expression of HLA class I, antigen presentation-related genes, and chemokines. NLRC5 also mediates the effects of IFNα on alternative splicing, a generator of ß cell neoantigens, suggesting that it is a central player of the effects of IFNα on ß cells that contribute to trigger and amplify autoimmunity in T1D.
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Diabetes Mellitus Tipo 1 , Células Secretoras de Insulina , Ilhotas Pancreáticas , Processamento Alternativo , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Humanos , Interferon-alfa/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ilhotas Pancreáticas/metabolismo , Transcrição GênicaRESUMO
BACKGROUND: Type 1 diabetes (T1D) is an autoimmune disease characterized by the progressive destruction of pancreatic beta cells. Interferon-α (IFNα), an antiviral cytokine, is expressed in the pancreatic islets in early T1D, which may be secondary to viral infections. However, not all patients harboring a type I IFN signature present signals of viral infection, suggesting that this response might be initiated by other "danger signals". Accumulation of mitochondrial double-stranded RNA (mtdsRNA; a danger signal), secondary to silencing of members of the mitochondrial degradosome, PNPT1 and SUV3, has been described to activate the innate immune response. METHODS: To evaluate whether mtdsRNA represents a "danger signal" for pancreatic beta cells in the context of T1D, we silenced PNPT1 and/or SUV3 in slowly proliferating human insulin-secreting EndoC-ßH1 cells and in non-proliferating primary human beta cells and evaluated dsRNA accumulation by immunofluorescence and the type I IFN response by western blotting and RT-qPCR. RESULTS: Only the simultaneous silencing of PNPT1/SUV3 induced dsRNA accumulation in EndoC-ßH1 cells but not in dispersed human islets, and there was no induction of a type I IFN response. By contrast, silencing of these two genes individually was enough to induce dsRNA accumulation in fibroblasts present in the human islet preparations. CONCLUSIONS: These data suggest that accumulation of endogenous mtdsRNA following degradosome knockdown depends on the proliferative capacity of the cells and is not a mediator of the type I IFN response in human pancreatic beta cells.
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Exposure of human pancreatic beta cells to pro-inflammatory cytokines or metabolic stressors is used to model events related to type 1 and type 2 diabetes, respectively. Quantitative real-time PCR is commonly used to quantify changes in gene expression. The selection of the most adequate reference gene(s) for gene expression normalization is an important pre-requisite to obtain accurate and reliable results. There are no universally applicable reference genes, and the human beta cell expression of commonly used reference genes can be altered by different stressors. Here we aimed to identify the most stably expressed genes in human beta cells to normalize quantitative real-time PCR gene expression.We used comprehensive RNA-sequencing data from the human pancreatic beta cell line EndoC-ßH1, human islets exposed to cytokines or the free fatty acid palmitate in order to identify the most stably expressed genes. Genes were filtered based on their level of significance (adjusted P-value >0.05), fold-change (|fold-change| <1.5) and a coefficient of variation <10%. Candidate reference genes were validated by quantitative real-time PCR in independent samples.We identified a total of 264 genes stably expressed in EndoC-ßH1 cells and human islets following cytokines - or palmitate-induced stress, displaying a low coefficient of variation. Validation by quantitative real-time PCR of the top five genes ARF1, CWC15, RAB7A, SIAH1 and VAPA corroborated their expression stability under most of the tested conditions. Further validation in independent samples indicated that the geometric mean of ACTB and VAPA expression can be used as a reliable normalizing factor in human beta cells.
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Genômica/métodos , Células Secretoras de Insulina , Humanos , Células Secretoras de Insulina/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In pancreatic ß-cells, the expression of the splicing factor SRSF6 is regulated by GLIS3, a transcription factor encoded by a diabetes susceptibility gene. SRSF6 down-regulation promotes ß-cell demise through splicing dysregulation of central genes for ß-cells function and survival, but how RNAs are targeted by SRSF6 remains poorly understood. Here, we define the SRSF6 binding landscape in the human pancreatic ß-cell line EndoC-ßH1 by integrating individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) under basal conditions with RNA sequencing after SRSF6 knockdown. We detect thousands of SRSF6 bindings sites in coding sequences. Motif analyses suggest that SRSF6 specifically recognizes a purine-rich consensus motif consisting of GAA triplets and that the number of contiguous GAA triplets correlates with increasing binding site strength. The SRSF6 positioning determines the splicing fate. In line with its role in ß-cell function, we identify SRSF6 binding sites on regulated exons in several diabetes susceptibility genes. In a proof-of-principle, the splicing of the susceptibility gene LMO7 is modulated by antisense oligonucleotides. Our present study unveils the splicing regulatory landscape of SRSF6 in immortalized human pancreatic ß-cells.
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Processamento Alternativo/genética , Regulação da Expressão Gênica , Células Secretoras de Insulina/metabolismo , Fosfoproteínas/metabolismo , RNA/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Sítios de Ligação , Linhagem Celular , Sobrevivência Celular/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 2/genética , Éxons , Técnicas de Silenciamento de Genes , Humanos , Proteínas com Domínio LIM/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de Proteínas , Fatores de Processamento de Serina-Arginina/química , Fatores de Processamento de Serina-Arginina/genética , Fatores de Transcrição/genética , Transcriptoma , TransfecçãoRESUMO
Interferon-α (IFNα), a type I interferon, is expressed in the islets of type 1 diabetic individuals, and its expression and signaling are regulated by T1D genetic risk variants and viral infections associated with T1D. We presently characterize human beta cell responses to IFNα by combining ATAC-seq, RNA-seq and proteomics assays. The initial response to IFNα is characterized by chromatin remodeling, followed by changes in transcriptional and translational regulation. IFNα induces changes in alternative splicing (AS) and first exon usage, increasing the diversity of transcripts expressed by the beta cells. This, combined with changes observed on protein modification/degradation, ER stress and MHC class I, may expand antigens presented by beta cells to the immune system. Beta cells also up-regulate the checkpoint proteins PDL1 and HLA-E that may exert a protective role against the autoimmune assault. Data mining of the present multi-omics analysis identifies two compound classes that antagonize IFNα effects on human beta cells.
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Processamento Alternativo , Células Secretoras de Insulina/fisiologia , Interferon-alfa/metabolismo , Interferon-alfa/farmacologia , Processamento Alternativo/efeitos dos fármacos , Células Cultivadas , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Mineração de Dados , Diabetes Mellitus Tipo 1/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Mapas de Interação de Proteínas , Proteômica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sítio de Iniciação de TranscriçãoRESUMO
INTRODUCTION: Erectile dysfunction (ED) is a common complication of diabetes. Endothelial cell (EC) dysfunction is one of the main mechanisms of diabetic ED. However, loss of EC integrity has never been assessed in human diabetic corpus cavernosum. AIM: To identify and quantify apoptotic cells in human diabetic and normal erectile tissue and to compare these results with each patient's clinical data and erection status. METHODS: Eighteen cavernosal samples were collected, 13 from diabetics with ED and 5 from nondiabetic individuals. Cavernosal structure and cell proliferation status were evaluated by immunohistochemistry. Tissue integrity was assessed by terminal transferase dUTP nick end labeling assay, an index of apoptotic cell density (ACD) established and compared with each patient age, type of diabetes, arterial risk factors number, arterial/veno-occlusive disease, response to intracavernous vasoactive injections (ICI), and penile nitric oxide release test (PNORT). MAIN OUTCOME MEASURES: Establish an index of ACD and correlate those results with patient clinical data. RESULTS: Nondiabetic samples presented few scattered cells in apoptosis and an ACD of 7.15 +/- 0.44 (mean apoptotic cells/tissue area mm(2) +/- standard error). The diabetic group showed an increased ACD of 23.82 +/- 1.53, and apoptotic cells were located specifically at vascular sites. Rehabilitation of these endothelial lesions seemed impaired, as no evidence of EC proliferation was observed. Furthermore, higher ACD in diabetic individuals correlated to poor response to PNORT and to ICI. CONCLUSIONS: We provided evidence for the first time that loss of cavernosal EC integrity is a crucial event involved in diabetic ED. Furthermore, we were able to establish a threshold between ACD values and cavernosal tissue functionality, as assessed by PNORT and vasoactive ICI.
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Apoptose/fisiologia , Diabetes Mellitus Tipo 2/epidemiologia , Endotélio Vascular/patologia , Disfunção Erétil/tratamento farmacológico , Disfunção Erétil/epidemiologia , Impotência Vasculogênica/tratamento farmacológico , Impotência Vasculogênica/epidemiologia , Vasodilatadores/uso terapêutico , Contagem de Células , Disfunção Erétil/diagnóstico , Humanos , Impotência Vasculogênica/diagnóstico , Masculino , Pessoa de Meia-Idade , Óxido Nítrico/administração & dosagem , Óxido Nítrico/farmacologia , Pênis/efeitos dos fármacos , Qualidade de Vida/psicologia , Fatores de RiscoRESUMO
BACKGROUND: Galectin-3 (Gal3) expression is associated with accumulation of Advanced Glycation End products (AGE), a common feature in diabetes mellitus (DM). The role of Gal3 in oxidative stress is, however, controversial, being considered in the literature to play either a protective role or exacerbating disease. METHODS: Herein, we examined the interplay between Gal3 and oxidative stress in a high-fat diet -induced type 2 DMC57Bl/6 mice model. Because natural polyphenols are known to play antioxidant and anti-inflammatory roles and to modulate metabolic activity, we further evaluated the effect of xanthohumol and 8-prenylnaringenin polyphenols in this crosstalk. RESULTS: Gal3 expression was accompanied by 3-nitrotyrosine and AGE production in liver and kidney of diabetic mice compared to healthy animals (fed with standard diet). Oral supplementation with polyphenols decreased the levels of these oxidative biomarkers as evaluated by immunohistochemistry and western blotting. Interestingly, blocking Gal3 by incubating human microvascular endothelial cells with modified citrus pectin increased 3-nitrotyrosine protein expression. CONCLUSIONS: These findings imply that Gal3 overexpression is probably controlling oxidative stress in endothelial cells. In conclusion, our results indicate that supplementation with 8-prenylnaringenin or xanthohumol reverses diabetes-associated oxidation in liver and kidney, and consequently decreases this diabetic biomarker that predispose to cardiovascular complications.
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BACKGROUND: Antibodies targeting PD-1 and its ligand PDL1 are used in cancer immunotherapy but may lead to autoimmune diseases, including type 1 diabetes (T1D). It remains unclear whether PDL1 is expressed in pancreatic islets of people with T1D and how is it regulated. METHODS: The expression of PDL1, IRF1, insulin and glucagon was evaluated in samples of T1D donors by immunofluorescence. Cytokine-induced PDL1 expression in the human beta cell line, EndoC-ßH1, and in primary human pancreatic islets was determined by real-time RT-PCR, flow cytometry and Western blot. Specific and previously validated small interference RNAs were used to inhibit STAT1, STAT2, IRF1 and JAK1 signaling. Key results were validated using the JAK inhibitor Ruxolitinib. FINDINGS: PDL1 was present in insulin-positive cells from twelve T1D individuals (6 living and 6 deceased donors) but absent from insulin-deficient islets or from the islets of six non-diabetic controls. Interferons-α and -γ, but not interleukin-1ß, induced PDL1 expression in vitro in human islet cells and EndoC-ßH1 cells. Silencing of STAT1 or STAT2 individually did not prevent interferon-α-induced PDL1, while blocking of JAKs - a proposed therapeutic strategy for T1D - or IRF1 prevented PDL1 induction. INTERPRETATION: These findings indicate that PDL1 is expressed in beta cells from people with T1D, possibly to attenuate the autoimmune assault, and that it is induced by both type I and II interferons via IRF1.
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Antígeno B7-H1/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Regulação da Expressão Gênica , Fator Regulador 1 de Interferon/metabolismo , Interferon-alfa/metabolismo , Interferon gama/metabolismo , Ilhotas Pancreáticas/metabolismo , Adolescente , Adulto , Biomarcadores , Linhagem Celular , Criança , Pré-Escolar , Humanos , Células Secretoras de Insulina/metabolismo , Pessoa de Meia-Idade , Adulto JovemRESUMO
Erectile dysfunction (ED) is a common complication of diabetes, affecting up to 75% of all diabetic men. Although the aetiology of diabetic ED is multifactorial, endothelial dysfunction is recognized as a mainstay in the pathophysiology of the disease. Endothelial dysfunction is induced by the detrimental actions of high glucose levels and increased oxidative stress on endothelial cells that make up the vascular lining. Besides directly injuring the endothelium, diabetes might also hamper vascular repair mechanisms of angiogenesis and vasculogenesis. These states exacerbate and maintain endothelial dysfunction, impairing vasorelaxation events and cavernosal blood perfusion, which are crucial for normal erectile function.
Assuntos
Diabetes Mellitus/sangue , Endotélio Vascular/metabolismo , Disfunção Erétil/sangue , Estresse Oxidativo/fisiologia , Ereção Peniana/fisiologia , Animais , Diabetes Mellitus/epidemiologia , Diabetes Mellitus/fisiopatologia , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Disfunção Erétil/epidemiologia , Disfunção Erétil/fisiopatologia , Humanos , MasculinoRESUMO
BACKGROUND: Erectile dysfunction (ED) is a prevalent complication of diabetes, and oxidative stress is an important feature of diabetic ED. Oxidative stress-induced damage plays a pivotal role in the development of tissue alterations. However, the deleterious effects of oxidative stress in the corpus cavernosum with the progression of diabetes remain unclear. The aim of this study was to evaluate systemic and penile oxidative stress status in the early and late stages of diabetes. METHODS: Male Wistar streptozotocin-diabetic rats (and age-matched controls) were examined 2 (early) and 8 weeks (late) after the induction of diabetes. Systemic oxidative stress was evaluated by urinary H2 O2 and the ratio of circulating reduced/oxidized glutathione (GSH/GSSG). Penile oxidative status was assessed by H2 O2 production and 3-nitrotyrosine (3-NT) formation. Cavernosal endothelial nitric oxide synthase (eNOS) was analyzed by quantitative immunohistochemistry. Dual immunofluorescence was also performed for 3-NT and α-smooth muscle actin (α-SMA) and eNOS-α-SMA. RESULTS: There was a significant increase in urinary H2 O2 levels in both diabetic groups. The plasma GSH/GSSG ratio was significantly augmented in late diabetes. In cavernosal tissue, H2 O2 production was significantly increased in late diabetes. Reactivity for 3-NT was located predominantly in cavernosal smooth muscle (SM) and was significantly reduced in late diabetes. Quantitative immunohistochemistry revealed a significant decrease in eNOS levels in cavernosal SM and endothelium in late diabetes. CONCLUSIONS: The findings indicate that the noxious effects of oxidative stress are more prominent in late diabetes. Increased penile protein oxidative modifications and decreased eNOS expression may be responsible for structural and/or functional deregulation, contributing to the progression of diabetes-associated ED.
Assuntos
Diabetes Mellitus Experimental/complicações , Endotélio Vascular/patologia , Disfunção Erétil/patologia , Miócitos de Músculo Liso/patologia , Óxido Nítrico Sintase Tipo III/metabolismo , Estresse Oxidativo , Pênis/patologia , Animais , Células Cultivadas , Diabetes Mellitus Experimental/patologia , Progressão da Doença , Endotélio Vascular/metabolismo , Disfunção Erétil/etiologia , Imunofluorescência , Immunoblotting , Masculino , Miócitos de Músculo Liso/metabolismo , Pênis/metabolismo , Ratos , Ratos Wistar , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
A body of growing evidence now implicates white adipose tissue as a relevant source of stromal progenitor cells recruited to the tumor microenvironment to form supportive tumor stroma. While the role of periprostatic (PP) adipose tissue in prostate cancer progression has been barely appreciated, we sought to determine the progenitor cell population in PP adipose tissue and the association with prostate cancer. We isolated and characterized CD31(-)CD34(+)CD45(-)CD146(-) progenitor cells (adipose-derived stem cells [ASC]) in paired samples of PP and preperitoneal visceral adipose tissue from prostate tissue and peripheral blood mononuclear cells of prostate cancer and nodular prostatic hyperplasia patients. ASC were quantified by flow cytometry and confirmed through target gene expression. Here we show a significantly higher amount of ASC in PP than in visceral adipose tissue, independent of body mass index and prostatic disease. In the prostate, ASC are increased in cancer compared with prostatic nodular hyperplasia patients. Concordantly, adipsin gene (CFD) expression, which is known to be up-regulated in adipose stem cells, was overexpressed in PP adipose tissue, in the prostate of cancer patients and in prostate CD31(-)CD34(+)CD45(-)CD146(-) sorted cells. ASC were found at higher levels in the blood of prostate cancer patients simultaneously overweight/obese. Present findings indicate that PP adipose tissue is a reservoir of progenitor cells with the potential to migrate towards prostate tumors, although its clinical significance merits further evaluation.