RESUMEN
OBJECTIVE: Distal symmetrical polyneuropathy (DSPN) is a severe common long-term complication of type 1 diabetes caused by impaired sensory-motor nerve function. As chronic low-grade inflammation may be involved in the pathogenesis of DSPN, we investigated the circulating levels of inflammatory markers in individuals with type 1 diabetes with and without DSPN. Furthermore, we determined to what extent these factors correlated with different peripheral sensory nerve functions. DESIGN: Cross-sectional study. PATIENTS: The study included 103 individuals with type 1 diabetes with (n = 50) and without DSPN (n = 53) as well as a cohort of healthy controls (n = 21). MEASUREMENTS: Circulating levels of various inflammatory markers (cytokines, chemokines and soluble adhesion molecules) were determined in serum samples by Luminex multiplexing technology. Peripheral sensory nerve testing, for example vibration, tactile and thermal perception, was assessed by standardized procedures. RESULTS: The cytokines IL-1α, IL-4, IL-12p70, IL-13, IL-17A and TNF-α; the chemokine MCP-1; and the adhesion molecule E-selectin were significantly increased in individuals with type 1 diabetes with DSPN compared to those without DSPN (P < .001). These observations were independent of age, sex, BMI, disease duration and blood pressure. Additionally, higher serum concentrations of cytokines and chemokines were associated with higher vibration and tactile perception thresholds, but not with heat tolerance threshold. CONCLUSIONS: Individuals with type 1 diabetes and concomitant DSPN display higher serum levels of several inflammatory markers. These findings support that systemic low-grade inflammation may play a role in the pathogenesis of DSPN.
Asunto(s)
Diabetes Mellitus Tipo 1 , Neuropatías Diabéticas , Polineuropatías , Biomarcadores , Estudios Transversales , Diabetes Mellitus Tipo 1/complicaciones , Humanos , Polineuropatías/etiologíaRESUMEN
INTRODUCTION: A neuroimmune communication exists, and compelling evidence suggests that diabetic neuropathy and systemic inflammation are linked. Our aims were (1) to investigate biomarkers of the ongoing inflammation processes including cytokines, adhesion molecules, and chemokines and (2) to associate the findings with cardiovascular autonomic neuropathy in type 1 diabetes by measuring heart rate variability and cardiac vagal tone. MATERIALS AND METHODS: We included 104 adults with type 1 diabetes. Heart rate variability, time domain, and frequency domains were calculated from a 24-hour Holter electrocardiogram, while cardiac vagal tone was determined from a 5-minute electrocardiogram. Cytokines (interleukin- (IL-) 1α, IL-4, IL-12p70, IL-13, IL-17, and tumor necrosis factor- (TNF-) α), adhesion molecules (E-selectin, P-selectin, and intercellular adhesion molecule- (ICAM-) 1), and chemokines (chemokine (C-C motif) ligand (CCL)2, CCL3, CCL4, and C-X-C motif chemokine (CXCL)10) were assessed using a Luminex multiplexing technology. Associations between concentrations of inflammatory biomarkers and continuous variables of heart rate variability and cardiac vagal tone were estimated using multivariable linear regression adjusting for age, sex, disease duration, and smoking. RESULTS: Participants with the presence of cardiovascular autonomic neuropathy had higher systemic levels of IL-1α, IL-4, CCL2, and E-selectin than those without cardiovascular autonomic neuropathy. IL-1α, IL-4, IL-12, TNF-α, and E-selectin were inversely associated with both sympathetic and parasympathetic heart rate variability measures (p > 0.01). Discussion. Our results show that several pro- and anti-inflammatory factors, believed to be involved in the progression of diabetic polyneuropathy, are associated with cardiovascular autonomic neuropathy, suggesting that these factors may also contribute to the pathogenesis of cardiovascular autonomic neuropathy. Our findings emphasize the importance of the neuroimmune regulatory system in the pathogenesis of neuropathy in type 1 diabetes.
Asunto(s)
Diabetes Mellitus Tipo 1/sangre , Frecuencia Cardíaca/fisiología , Inflamación/sangre , Adulto , Sistema Nervioso Autónomo , Biomarcadores , Quimiocinas/metabolismo , Quimiotaxis , Citocinas/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Reproducibilidad de los ResultadosRESUMEN
AIMS: To compare the effects of a low carbohydrate diet (LCD < 100 g carbohydrate/d) and a high carbohydrate diet (HCD > 250 g carbohydrate/d) on glycaemic control and cardiovascular risk factors in adults with type 1 diabetes. MATERIALS AND METHODS: In a randomized crossover study with two 12-week intervention arms separated by a 12-week washout, 14 participants using sensor-augmented insulin pumps were included. Individual meal plans meeting the carbohydrate criteria were made for each study participant. Actual carbohydrate intake was entered into the insulin pumps throughout the study. RESULTS: Ten participants completed the study. Daily carbohydrate intake during the two intervention periods was (mean ± standard deviation) 98 ± 11 g and 246 ± 34 g, respectively. Time spent in the range 3.9-10.0 mmol/L (primary outcome) did not differ between groups (LCD 68.6 ± 8.9% vs. HCD 65.3 ± 6.5%, P = 0.316). However, time spent <3.9 mmol/L was less (1.9 vs. 3.6%, P < 0.001) and glycaemic variability (assessed by coefficient of variation) was lower (32.7 vs. 37.5%, P = 0.013) during LCD. No events of severe hypoglycaemia were reported. Participants lost 2.0 ± 2.1 kg during LCD and gained 2.6 ± 1.8 kg during HCD (P = 0.001). No other cardiovascular risk factors, including fasting levels of lipids and inflammatory markers, were significantly affected. CONCLUSIONS: Compared with an intake of 250 g of carbohydrate per day, restriction of carbohydrate intake to 100 g per day in adults with type 1 diabetes reduced time spent in hypoglycaemia, glycaemic variability and weight with no effect on cardiovascular risk factors.
Asunto(s)
Diabetes Mellitus Tipo 1/dietoterapia , Dieta Baja en Carbohidratos , Adulto , Glucemia/análisis , Estudios Cruzados , Carbohidratos de la Dieta/administración & dosificación , Femenino , Humanos , Lipoproteínas/sangre , Masculino , Persona de Mediana Edad , Pérdida de Peso/fisiologíaRESUMEN
Over 40 susceptibility loci have been identified for type 1 diabetes (T1D). Little is known about how these variants modify disease risk and progression. Here, we combined in vitro and in vivo experiments with clinical studies to determine how genetic variation of the candidate gene cathepsin H (CTSH) affects disease mechanisms and progression in T1D. The T allele of rs3825932 was associated with lower CTSH expression in human lymphoblastoid cell lines and pancreatic tissue. Proinflammatory cytokines decreased the expression of CTSH in human islets and primary rat ß-cells, and overexpression of CTSH protected insulin-secreting cells against cytokine-induced apoptosis. Mechanistic studies indicated that CTSH exerts its antiapoptotic effects through decreased JNK and p38 signaling and reduced expression of the proapoptotic factors Bim, DP5, and c-Myc. CTSH overexpression also up-regulated Ins2 expression and increased insulin secretion. Additionally, islets from Ctsh(-/-) mice contained less insulin than islets from WT mice. Importantly, the TT genotype was associated with higher daily insulin dose and faster disease progression in newly diagnosed T1D patients, indicating agreement between the experimental and clinical data. In line with these observations, healthy human subjects carrying the T allele have lower ß-cell function, which was evaluated by glucose tolerance testing. The data provide strong evidence that CTSH is an important regulator of ß-cell function during progression of T1D and reinforce the concept that candidate genes for T1D may affect disease progression by modulating survival and function of pancreatic ß-cells, the target cells of the autoimmune assault.
Asunto(s)
Catepsina H/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Células Secretoras de Insulina/metabolismo , Adolescente , Alelos , Animales , Apoptosis/genética , Catepsina H/genética , Línea Celular , Niño , Preescolar , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 1/terapia , Regulación de la Expresión Génica/genética , Genotipo , Humanos , Células Secretoras de Insulina/patología , Ratones , Ratones Noqueados , RatasRESUMEN
Type 1 diabetes (T1D) is a multifactorial disease resulting from an immune-mediated destruction of the insulin-producing pancreatic ß cells. Several environmental and genetic risk factors predispose to the disease. Genome-wide association studies (GWAS) have identified around 50 genetic regions that affect the risk of developing T1D, but the disease-causing variants and genes are still largely unknown. In this review, we discuss the current status of T1D susceptibility loci and candidate genes with focus on the ß cell. At least 40 % of the genes in the T1D susceptibility loci are expressed in human islets and ß cells, where they according to recent studies modulate the ß-cell response to the immune system. As most of the risk variants map to noncoding regions of the genome, i.e., promoters, enhancers, intergenic regions, and noncoding genes, their possible involvement in T1D pathogenesis as gene regulators will also be addressed.
Asunto(s)
Diabetes Mellitus Tipo 1/genética , Regulación de la Expresión Génica , Células Secretoras de Insulina/metabolismo , Animales , Estudio de Asociación del Genoma Completo , Humanos , Insulina/genética , Células Secretoras de Insulina/patología , ARN no Traducido/genéticaRESUMEN
Accumulating data suggest a role for the lysosomal protease cathepsin S (CTSS) in type 1 diabetes. Circulating CTSS is increased in type 1 diabetes; however, whether CTSS has protective or deleterious effects is unclear. The study's objectives were to examine the biomarker potential of CTSS in new-onset type 1 diabetes, and to investigate the expression and secretion of CTSS in human islets and ß-cells. The CTSS level was analyzed in serum from children with new-onset type 1 diabetes and autoantibody-positive and -negative siblings by ELISA. The expression and secretion of CTSS were evaluated in isolated human islets and EndoC-ßH5 cells by real-time qPCR, immunoblotting, and ELISA. The CTSS serum level was elevated in children with new-onset type 1 diabetes and positively associated with autoantibody status in healthy siblings. Human islets and EndoC-ßH5 cells demonstrated induction and secretion of CTSS after exposure to proinflammatory cytokines, a model system of islet inflammation. Analysis of publicly available single-cell RNA sequencing data on human islets showed that elevated CTSS expression was exclusive for the ß-cells in donors with type 1 diabetes as compared with nondiabetic donors. These findings suggest a potential of CTSS as a diagnostic biomarker in type 1 diabetes.
Asunto(s)
Autoanticuerpos , Catepsinas , Diabetes Mellitus Tipo 1 , Islotes Pancreáticos , Hermanos , Humanos , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/sangre , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Catepsinas/sangre , Masculino , Niño , Femenino , Islotes Pancreáticos/inmunología , Adolescente , Células Secretoras de Insulina/inmunología , Células Secretoras de Insulina/metabolismo , Preescolar , Biomarcadores/sangreRESUMEN
Objective: EndoC-ßH5 is a newly established human beta-cell model which may be superior to previous model systems. Exposure of beta cells to pro-inflammatory cytokines is widely used when studying immune-mediated beta-cell failure in type 1 diabetes. We therefore performed an in-depth characterization of the effects of cytokines on EndoC-ßH5 cells. Methods: The sensitivity profile of EndoC-ßH5 cells to the toxic effects of interleukin-1ß (IL-1ß), interferon γ (IFNγ) and tumor necrosis factor-α (TNFα) was examined in titration and time-course experiments. Cell death was evaluated by caspase-3/7 activity, cytotoxicity, viability, TUNEL assay and immunoblotting. Activation of signaling pathways and major histocompatibility complex (MHC)-I expression were examined by immunoblotting, immunofluorescence, and real-time quantitative PCR (qPCR). Insulin and chemokine secretion were measured by ELISA and Meso Scale Discovery multiplexing electrochemiluminescence, respectively. Mitochondrial function was evaluated by extracellular flux technology. Global gene expression was characterized by stranded RNA sequencing. Results: Cytokines increased caspase-3/7 activity and cytotoxicity in EndoC-ßH5 cells in a time- and dose-dependent manner. The proapoptotic effect of cytokines was primarily driven by IFNγ signal transduction. Cytokine exposure induced MHC-I expression and chemokine production and secretion. Further, cytokines caused impaired mitochondrial function and diminished glucose-stimulated insulin secretion. Finally, we report significant changes to the EndoC-ßH5 transcriptome including upregulation of the human leukocyte antigen (HLA) genes, endoplasmic reticulum stress markers, and non-coding RNAs, in response to cytokines. Among the differentially expressed genes were several type 1 diabetes risk genes. Conclusion: Our study provides detailed insight into the functional and transcriptomic effects of cytokines on EndoC-ßH5 cells. This information should be useful for future studies using this novel beta-cell model.
Asunto(s)
Citocinas , Diabetes Mellitus Tipo 1 , Humanos , Transcriptoma , Caspasa 3/genética , Interferón gamma/farmacología , QuimiocinasRESUMEN
Pancreatic ß cells are central to glycemic regulation through insulin production. Studies show autophagy as an essential process in ß cell function and fate. Autophagy is a catabolic cellular process that regulates cell homeostasis by recycling surplus or damaged cell components. Impaired autophagy results in ß cell loss of function and apoptosis and, as a result, diabetes initiation and progress. It has been shown that in response to endoplasmic reticulum stress, inflammation, and high metabolic demands, autophagy affects ß cell function, insulin synthesis, and secretion. This review highlights recent evidence regarding how autophagy can affect ß cells' fate in the pathogenesis of diabetes. Furthermore, we discuss the role of important intrinsic and extrinsic autophagy modulators, which can lead to ß cell failure.
Asunto(s)
Diabetes Mellitus , Células Secretoras de Insulina , Humanos , Células Secretoras de Insulina/metabolismo , Diabetes Mellitus/metabolismo , Insulina/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Autofagia/fisiologíaRESUMEN
Circular RNAs (circRNAs) have recently been implicated in impaired ß-cell function in diabetes. Using microarray-based profiling of circRNAs in human EndoC-ßH1 cells treated with pro-inflammatory cytokines, this study aimed to investigate the expression and possible regulatory roles of circRNAs in human ß cells. We identified ~5000 ß-cell-expressed circRNAs, of which 84 were differentially expressed (DE) after cytokine exposure. Pathway analysis of the host genes of the DE circRNAs revealed the enrichment of cytokine signaling pathways, indicative of circRNA transcription from inflammatory genes in response to cytokines. Multiple binding sites for ß-cell-enriched microRNAs and RNA-binding proteins were observed for the highly upregulated circRNAs, supporting their function as 'sponges' or 'decoys'. We also present evidence for circRNA sequence conservation in multiple species, the presence of cytokine-induced regulatory elements, and putative protein-coding potential for the DE circRNAs. This study highlights the complex regulatory potential of circRNAs, which may play a crucial role during immune-mediated ß-cell destruction in type 1 diabetes.
RESUMEN
BACKGROUND: Several approaches have been developed for miRNA target prediction, including methods that incorporate expression profiling. However the methods are still in need of improvements due to a high false discovery rate. So far, none of the methods have used independent component analysis (ICA). Here, we developed a novel target prediction method based on ICA that incorporates both seed matching and expression profiling of miRNA and mRNA expressions. The method was applied on a cellular model of type 1 diabetes. RESULTS: Microarray profiling identified eight miRNAs (miR-124/128/192/194/204/375/672/708) with differential expression. Applying ICA on the mRNA profiling data revealed five significant independent components (ICs) correlating to the experimental conditions. The five ICs also captured the miRNA expressions by explaining > 97% of their variance. By using ICA, seven of the eight miRNAs showed significant enrichment of sequence predicted targets, compared to only four miRNAs when using simple negative correlation. The ICs were enriched for miRNA targets that function in diabetes-relevant pathways e.g. type 1 and type 2 diabetes and maturity onset diabetes of the young (MODY). CONCLUSIONS: In this study, ICA was applied as an attempt to separate the various factors that influence the mRNA expression in order to identify miRNA targets. The results suggest that ICA is better at identifying miRNA targets than negative correlation. Additionally, combining ICA and pathway analysis constitutes a means for prioritizing between the predicted miRNA targets. Applying the method on a model of type 1 diabetes resulted in identification of eight miRNAs that appear to affect pathways of relevance to disease mechanisms in diabetes.
Asunto(s)
Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , MicroARNs/genética , Western Blotting , Línea Celular , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Emerging evidence suggests that several of the lysosomal cathepsin proteases are genetically associated with type 1 diabetes (T1D) and participate in immune-mediated destruction of the pancreatic ß cells. We previously reported that the T1D candidate gene cathepsin H is downregulated by pro-inflammatory cytokines in human pancreatic islets and regulates ß-cell function, apoptosis, and disease progression in children with new-onset T1D. In the present study, the objective was to investigate the expression patterns of all 15 known cathepsins in ß-cell model systems and examine their role in the regulation of cytokine-induced apoptosis. Real-time qPCR screening of the cathepsins in human islets, 1.1B4 and INS-1E ß-cell models identified several cathepsins that were expressed and regulated by pro-inflammatory cytokines. Using small interfering RNAs to knock down (KD) the cytokine-regulated cathepsins, we identified an anti-apoptotic function of cathepsin C as KD increased cytokine-induced apoptosis. KD of cathepsin C correlated with increased phosphorylation of JNK and p38 mitogen-activated protein kinases, and elevated chemokine CXCL10/IP-10 expression. This study suggests that cathepsin C is a modulator of ß-cell survival, and that immune modulation of cathepsin expression in islets may contribute to immune-mediated ß-cell destruction in T1D.
Asunto(s)
Apoptosis , Catepsina C/fisiología , Citocinas/farmacología , Células Secretoras de Insulina , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Catepsina C/genética , Células Cultivadas , Citocinas/metabolismo , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patología , Humanos , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/patología , Células Secretoras de Insulina/fisiología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/patología , Islotes Pancreáticos/fisiología , Modelos Biológicos , RatasRESUMEN
The single nucleotide polymorphism rs7804356 located in the Src kinase-associated phosphoprotein 2 (SKAP2) gene is associated with type 1 diabetes (T1D), suggesting SKAP2 as a causal candidate gene. The objective of the study was to investigate if SKAP2 has a functional role in the ß-cells in relation to T1D. In a cohort of children with newly diagnosed T1D, rs7804356 predicted glycemic control and residual ß-cell function during the 1st year after diagnosis. In INS-1E cells and rat and human islets, proinflammatory cytokines reduced the content of SKAP2. Functional studies revealed that knockdown of SKAP2 aggravated cytokine-induced apoptosis in INS-1E cells and primary rat ß-cells, suggesting an antiapoptotic function of SKAP2. In support of this, overexpression of SKAP2 afforded protection against cytokine-induced apoptosis, which correlated with reduced nuclear content of S536-phosphorylated nuclear factor-κB (NF-κB) subunit p65, lower nitric oxide production, and diminished CHOP expression indicative of decreased endoplasmic reticulum stress. Knockdown of CHOP partially counteracted the increase in cytokine-induced apoptosis caused by SKAP2 knockdown. In conclusion, our results suggest that SKAP2 controls ß-cell sensitivity to cytokines possibly by affecting the NF-κB-inducible nitric oxide synthase-endoplasmic reticulum stress pathway.
Asunto(s)
Apoptosis/genética , Glucemia/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Células Secretoras de Insulina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Polimorfismo de Nucleótido Simple , Adolescente , Animales , Glucemia/genética , Niño , Preescolar , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/genética , Femenino , Técnicas de Silenciamiento del Gen , Genotipo , Control Glucémico , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Islotes Pancreáticos/metabolismo , Masculino , RatasRESUMEN
The type 1 diabetes (T1D) risk locus on chromosome 15q25.1 harbors the candidate gene CTSH (cathepsin H). We previously demonstrated that CTSH regulates ß-cell function in vitro and in vivo. CTSH overexpression protected insulin-secreting INS-1 cells against cytokine-induced apoptosis. The purpose of the present study was to identify the genes through which CTSH mediates its protective effects. Microarray analysis identified 63 annotated genes differentially expressed between CTSH-overexpressing INS-1 cells and control cells treated with interleukin-1ß and interferon-γ for up to 16h. Permutation test identified 10 significant genes across all time-points: Elmod1, Fam49a, Gas7, Gna15, Msrb3, Nox1, Ptgs1, Rac2, Scn7a and Ttn. Pathway analysis identified the "Inflammation mediated by chemokine and cytokine signaling pathway" with Gna15, Ptgs1 and Rac2 as significant. Knockdown of Rac2 abolished the protective effect of CTSH overexpression on cytokine-induced apoptosis, suggesting that the small GTPase and T1D candidate gene Rac2 contributes to the anti-apoptotic effect of CTSH.
Asunto(s)
Apoptosis , Catepsina H/genética , Citocinas/farmacología , Células Secretoras de Insulina/fisiología , Proteínas de Unión al GTP rac/fisiología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Catepsina H/fisiología , Células Cultivadas , Citoprotección/efectos de los fármacos , Citoprotección/genética , Humanos , Células Secretoras de Insulina/efectos de los fármacos , Ratones , Ratas , Proteína RCA2 de Unión a GTPRESUMEN
Inflammatory ß-cell failure contributes to type 1 and type 2 diabetes pathogenesis. Pro-inflammatory cytokines cause ß-cell dysfunction and apoptosis, and lysine deacetylase inhibitors (KDACi) prevent ß-cell failure in vitro and in vivo, in part by reducing NF-κB transcriptional activity. We investigated the hypothesis that the protective effect of KDACi involves transcriptional regulation of microRNAs (miRs), potential new targets in diabetes treatment. Insulin-producing INS1 cells were cultured with or without the broad-spectrum KDACi Givinostat, prior to exposure to the pro-inflammatory cytokines IL-1ß and IFN-γ for 6 h or 24 h, and miR expression was profiled with miR array. Thirteen miRs (miR-7a-2-3p, miR-29c-3p, miR-96-5p, miR-101a-3p, miR-140-5p, miR-146a-5p, miR-146b-5p, miR-340-5p, miR-384-5p, miR-455-5p, miR-466b-2-3p, miR-652-5p, and miR-3584-5p) were regulated by both cytokines and Givinostat, and nine were examined by qRT-PCR. miR-146a-5p was strongly regulated by cytokines and KDACi and was analyzed further. miR-146a-5p expression was induced by cytokines in rat and human islets. Cytokine-induced miR-146a-5p expression was specific for INS1 and ß-TC3 cells, whereas α-TC1 cells exhibited a higher basal expression. Transfection of INS1 cells with miR-146a-5p reduced cytokine signaling, including the activity of NF-κB and iNOS promoters, as well as NO production and protein levels of iNOS and its own direct targets TNF receptor associated factor 6 (TRAF6) and interleukin-1 receptor-associated kinase 1 (IRAK1). miR-146a-5p was elevated in the pancreas of diabetes-prone BB-DP rats at diabetes onset, suggesting that miR-146a-5p could play a role in type 1 diabetes development. The miR array of cytokine-exposed INS1 cells rescued by KDACi revealed several other miRs potentially involved in cytokine-induced ß-cell apoptosis, demonstrating the strength of this approach.
Asunto(s)
Diabetes Mellitus/genética , Inhibidores de Histona Desacetilasas/farmacología , Células Secretoras de Insulina/fisiología , MicroARNs/fisiología , Adulto , Animales , Apoptosis , Línea Celular , Citocinas/metabolismo , Diabetes Mellitus/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Células Secretoras de Insulina/citología , Islotes Pancreáticos/metabolismo , Masculino , Persona de Mediana Edad , FN-kappa B/genética , FN-kappa B/metabolismo , Ratas , Ratas WistarRESUMEN
The study aimed to explore the role of ERBB3 in type 1 diabetes (T1D). We examined whether genetic variation of ERBB3 (rs2292239) affects residual ß-cell function in T1D cases. Furthermore, we examined the expression of ERBB3 in human islets, the effect of ERBB3 knockdown on apoptosis in insulin-producing INS-1E cells and the genetic and regulatory architecture of the ERBB3 locus to provide insights to how rs2292239 may confer disease susceptibility. rs2292239 strongly correlated with residual ß-cell function and metabolic control in children with T1D. ERBB3 locus associated lncRNA (NONHSAG011351) was found to be expressed in human islets. ERBB3 was expressed and down-regulated by pro-inflammatory cytokines in human islets and INS-1E cells; knockdown of ERBB3 in INS-1E cells decreased basal and cytokine-induced apoptosis. Our data suggests an important functional role of ERBB3 and its potential regulators in the ß-cells and may constitute novel targets to prevent ß-cell destruction in T1D.
Asunto(s)
Diabetes Mellitus Tipo 1/genética , Polimorfismo de Nucleótido Simple , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismo , Animales , Apoptosis , Células Cultivadas , Niño , Estudios Transversales , Citocinas/genética , Diabetes Mellitus Tipo 1/inmunología , Femenino , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Masculino , ARN Largo no Codificante/genética , RatasRESUMEN
Pancreatic ß-cells are destroyed by an autoimmune attack in type 1 diabetes. Linkage and genome-wide association studies point to >50 loci that are associated with the disease in the human genome. Pathway analysis of candidate genes expressed in human islets identified a central role for interferon (IFN)-regulated pathways and tyrosine kinase 2 (TYK2). Polymorphisms in the TYK2 gene predicted to decrease function are associated with a decreased risk of developing type 1 diabetes. We presently evaluated whether TYK2 plays a role in human pancreatic ß-cell apoptosis and production of proinflammatory mediators. TYK2-silenced human ß-cells exposed to polyinosinic-polycitidilic acid (PIC) (a mimick of double-stranded RNA produced during viral infection) showed less type I IFN pathway activation and lower production of IFNα and CXCL10. These cells also had decreased expression of major histocompatibility complex (MHC) class I proteins, a hallmark of early ß-cell inflammation in type 1 diabetes. Importantly, TYK2 inhibition prevented PIC-induced ß-cell apoptosis via the mitochondrial pathway of cell death. The present findings suggest that TYK2 regulates apoptotic and proinflammatory pathways in pancreatic ß-cells via modulation of IFNα signaling, subsequent increase in MHC class I protein, and modulation of chemokines such as CXCL10 that are important for recruitment of T cells to the islets.
Asunto(s)
Apoptosis/genética , Diabetes Mellitus Tipo 1/genética , Inmunidad Innata/genética , Células Secretoras de Insulina/metabolismo , TYK2 Quinasa/genética , Apoptosis/inmunología , Línea Celular , Supervivencia Celular/genética , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Genes MHC Clase I/fisiología , Estudio de Asociación del Genoma Completo , Humanos , Células Secretoras de Insulina/inmunología , Interferón-alfa/genética , Interferón-alfa/metabolismo , Fosforilación , Polimorfismo de Nucleótido Simple , TYK2 Quinasa/metabolismoRESUMEN
Transcription factor CEBPA has been widely studied for its involvement in hematopoietic cell differentiation and causal role in hematological malignancies. We demonstrate here that it also performs a causal role in cytokine-induced apoptosis of pancreas ß cells. Treatment of two mouse pancreatic α and ß cell lines (αTC1-6 and ßTC1) with proinflammatory cytokines IL-1ß, IFN-γ, and TNF-α at doses that specifically induce apoptosis of ßTC1 significantly increased the amount of mRNA and protein encoded by Cebpa and its proapoptotic targets, Arl6ip5 and Tnfrsf10b, in ßTC1 but not in αTC1-6. Cebpa knockdown in ßTC1 significantly decreased cytokine-induced apoptosis, together with the amount of Arl6ip5 and Tnfrsf10b. Analysis of the network comprising CEBPA, its targets, their first interactants, and proteins encoded by genes known to regulate cytokine-induced apoptosis in pancreatic ß cells (genes from the apoptotic machinery and from MAPK and NFkB pathways) revealed that CEBPA, ARL6IP5, TNFRSF10B, TRAF2, and UBC are the top five central nodes. In silico analysis further suggests TRAF2 as trait d'union node between CEBPA and the NFkB pathway. Our results strongly suggest that Cebpa is a key regulator within the apoptotic network activated in pancreatic ß cells during insulitis, and Arl6ip5, Tnfrsf10b, Traf2, and Ubc are key executioners of this program.
Asunto(s)
Apoptosis , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Células Secretoras de Insulina/metabolismo , Transducción de Señal , Animales , Proteínas Potenciadoras de Unión a CCAAT/genética , Línea Celular , Interferón gamma/farmacología , Interleucina-1beta/farmacología , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Genome-wide association studies (GWAS) have heralded a new era in susceptibility locus discovery in complex diseases. For type 1 diabetes, >40 susceptibility loci have been discovered. However, GWAS do not inevitably lead to identification of the gene or genes in a given locus associated with disease, and they do not typically inform the broader context in which the disease genes operate. Here, we integrated type 1 diabetes GWAS data with protein-protein interactions to construct biological networks of relevance for disease. A total of 17 networks were identified. To prioritize and substantiate these networks, we performed expressional profiling in human pancreatic islets exposed to proinflammatory cytokines. Three networks were significantly enriched for cytokine-regulated genes and, thus, likely to play an important role for type 1 diabetes in pancreatic islets. Eight of the regulated genes (CD83, IFNGR1, IL17RD, TRAF3IP2, IL27RA, PLCG2, MYO1B, and CXCR7) in these networks also harbored single nucleotide polymorphisms nominally associated with type 1 diabetes. Finally, the expression and cytokine regulation of these new candidate genes were confirmed in insulin-secreting INS-1 ß-cells. Our results provide novel insight to the mechanisms behind type 1 diabetes pathogenesis and, thus, may provide the basis for the design of novel treatment strategies.