RESUMEN
Caspase 1 is part of the inflammasome, which is assembled upon pathogen recognition, while caspases 3 and/or 7 are mediators of apoptotic and nonapoptotic functions. PARP1 cleavage is a hallmark of apoptosis yet not essential, suggesting it has another physiological role. Here we show that after LPS stimulation, caspase 7 is activated by caspase 1, translocates to the nucleus, and cleaves PARP1 at the promoters of a subset of NF-κB target genes negatively regulated by PARP1. Mutating the PARP1 cleavage site D214 renders PARP1 uncleavable and inhibits PARP1 release from chromatin and chromatin decondensation, thereby restraining the expression of cleavage-dependent NF-κB target genes. These findings propose an apoptosis-independent regulatory role for caspase 7-mediated PARP1 cleavage in proinflammatory gene expression and provide insight into inflammasome signaling.
Asunto(s)
Caspasa 7/fisiología , FN-kappa B/metabolismo , Poli(ADP-Ribosa) Polimerasas/fisiología , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas Portadoras/fisiología , Cromatina/metabolismo , Regulación de la Expresión Génica , Humanos , Inflamación/genética , Ratones , Mutación , Proteína con Dominio Pirina 3 de la Familia NLR , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/química , Poli(ADP-Ribosa) Polimerasas/genética , Transducción de SeñalRESUMEN
One of the fastest cellular responses to genotoxic stress is the formation of poly(ADP-ribose) polymers (PAR) by poly(ADP-ribose)polymerase 1 (PARP1, or ARTD1). PARP1 and its enzymatic product PAR regulate diverse biological processes, such as DNA repair, chromatin remodeling, transcription and cell death. However, the inter-dependent function of the PARP1 protein and its enzymatic activity clouds the mechanism underlying the biological response. We generated a PARP1 knock-in mouse model carrying a point mutation in the catalytic domain of PARP1 (D993A), which impairs the kinetics of the PARP1 activity and the PAR chain complexity in vitro and in vivo, designated as hypo-PARylation. PARP1D993A/D993A mice and cells are viable and show no obvious abnormalities. Despite a mild defect in base excision repair (BER), this hypo-PARylation compromises the DNA damage response during DNA replication, leading to cell death or senescence. Strikingly, PARP1D993A/D993A mice are hypersensitive to alkylation in vivo, phenocopying the phenotype of PARP1 knockout mice. Our study thus unravels a novel regulatory mechanism, which could not be revealed by classical loss-of-function studies, on how PAR homeostasis, but not the PARP1 protein, protects cells and organisms from acute DNA damage.
Asunto(s)
Daño del ADN , Células Madre Embrionarias de Ratones/metabolismo , Poli ADP Ribosilación , Poli(ADP-Ribosa) Polimerasas/metabolismo , Animales , Dominio Catalítico/genética , Células Cultivadas , Reparación del ADN , Replicación del ADN/genética , Cinética , Ratones , Ratones de la Cepa 129 , Ratones Transgénicos , Modelos Genéticos , Mutación , Poli(ADP-Ribosa) Polimerasas/genéticaRESUMEN
ADP-ribosyltransferase Diphtheria toxin-like 1 [ARTD1; formerly called poly-ADP-ribose polymerase 1 (PARP1)] is a chromatin-associated enzyme involved in regulating metabolic homeostasis. The liver is at the core of glucose and lipid metabolism and is significantly affected by obesity and the metabolic syndrome. Here, we show that when fed a high-fat diet (HFD), mice lacking ARTD1 developed exacerbated hepatic steatosis. ARTD1(-/-) mice had a 19% higher liver weight than wild-type (WT) animals and exhibited a significantly increased serum concentration of cholesterol (38%) and impaired glucose tolerance. In addition, adipocyte function and size were significantly reduced in ARTD1(-/-) mice fed an HFD (7794 µm(2) for WT and 5579 µm(2) for ARTD1(-/-) mice). The significantly reduced adipogenic differentiation of adipose-derived stromal cells (ASCs) isolated from ARTD1(-/-) mice (28 vs. 11% Oil red O-positive cells in WT and ARTD1(-/-) ASCs, respectively) suggested that impaired adipogenesis as the underlying cause for this adipose tissue malfunction. This function of ARTD1 was specific for adipogenesis, since osteogenic differentiation was not affected by the ARTD1 deletion. In summary, we show that ARTD1(-/-) mice fed an HFD display impaired adipogenesis and show exacerbated hepatic steatosis, which can have important implications for nonalcoholic fatty liver disease.
Asunto(s)
Hígado Graso/etiología , Hígado/metabolismo , Poli(ADP-Ribosa) Polimerasas/genética , Adipocitos/metabolismo , Adipogénesis , Tejido Adiposo/metabolismo , Animales , Diferenciación Celular , Colesterol/sangre , Dieta Alta en Grasa , Intolerancia a la Glucosa/etiología , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico , Osteogénesis , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/deficienciaRESUMEN
NF-κB regulates the expression of a large number of target genes involved in the immune and inflammatory response, apoptosis, cell proliferation, differentiation and survival. In this study, we identified SIRT2 as a deacetylase of the transcription factor p65. SIRT2 is a member of the family of sirtuins, which are NAD(+)-dependent deacetylases involved in several cellular processes. SIRT2 interacts with p65 in the cytoplasm and deacetylates p65 in vitro and in vivo at Lys310. Moreover, p65 is hyperacetylated at Lys310 in Sirt2(-/-) cells after TNFα stimulation, which results in the increase in expression of a subset of p65 acetylation-dependent target genes. Our work provides evidence that p65 is deacetylated by SIRT2 in the cytoplasm to regulate the expression of specific NF-κB-dependent genes.
Asunto(s)
Regulación de la Expresión Génica , Lisina/metabolismo , Sirtuina 2/metabolismo , Factor de Transcripción ReIA/metabolismo , Acetilación/efectos de los fármacos , Animales , Especificidad de Anticuerpos/efectos de los fármacos , Apoptosis/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Embrión de Mamíferos/citología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Cinética , Ratones , Células 3T3 NIH , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Reproducibilidad de los Resultados , Sirtuina 1/deficiencia , Sirtuina 1/metabolismo , Sirtuina 2/deficiencia , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genética , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Overconsumption of saturated fats promotes obesity and type 2 diabetes. Excess weight gain in early life may be particularly detrimental by promoting earlier diabetes onset and potentially by adversely affecting normal development. In the present study we investigated the effects of dietary fat composition on early overnutrition-induced body weight and glucose regulation in Swiss Webster mice, which show susceptibility to high-fat diet-induced diabetes. We compared glucose homeostasis between a high-fat lard-based (HFL) diet, high in saturated fats, and a high-fat olive oil/fish oil-based (HFO) diet, high in monounsaturated and omega-3 fats. We hypothesized that the healthier fat profile of the latter diet would improve early overnutrition-induced glucose dysregulation. However, early overnutrition HFO pups gained more weight and adiposity and had higher diabetes incidence compared to HFL. In contrast, control pups had less weight gain, adiposity, and lower diabetes incidence. Plasma metabolomics revealed reductions in various phosphatidylcholine species in early overnutrition HFO mice as well as with diabetes. These findings suggest that early overnutrition may negate any beneficial effects of a high-fat diet that favours monounsaturated and omega-3 fats over saturated fats. Thus, quantity, quality, and timing of fat intake throughout life should be considered with respect to metabolic health outcomes.
Asunto(s)
Dieta Alta en Grasa , Grasas Insaturadas en la Dieta/metabolismo , Metabolismo Energético , Ácidos Grasos Omega-3/metabolismo , Hipernutrición/metabolismo , Factores de Edad , Animales , Biomarcadores , Diabetes Mellitus Experimental , Glucosa/metabolismo , Hormonas/sangre , Hormonas/metabolismo , Células Secretoras de Insulina/metabolismo , Masculino , Ratones , Fosfatidilcolinas/sangreRESUMEN
Although innate immunity is linked to metabolic health, the effect of leptin signaling in cells from the innate immune system on glucose homeostasis has not been thoroughly investigated. We generated two mouse models using Cre-lox methodology to determine the effect of myeloid cell-specific leptin receptor (Lepr) reconstitution and Lepr knockdown on in vivo glucose metabolism. Male mice with myeloid cell-specific Lepr reconstitution (Lyz2Cre+LeprloxTB/loxTB) had better glycemic control as they aged compared to male mice with whole-body transcriptional blockade of Lepr (Lyz2Cre-LeprloxTB/loxTB). In contrast, Lyz2Cre+LeprloxTB/loxTB females only had a trend for diminished hyperglycemia after a prolonged fast. During glucose tolerance tests, Lyz2Cre+LeprloxTB/loxTB males had a mildly improved plasma glucose profile compared to Cre- controls while Lyz2Cre+LeprloxTB/loxTB females had a similar glucose excursion to their Cre- controls. Myeloid cell-specific Lepr knockdown (Lyz2Cre+Leprflox/flox) did not significantly alter body weight, blood glucose, insulin sensitivity, or glucose tolerance in males or females. Expression of the cytokine interleukin 10 (anti-inflammatory) tended to be higher in adipose tissue of male Lyz2Cre+LeprloxTB/loxTB mice (p = 0.0774) while interleukin 6 (pro-inflammatory) was lower in male Lyz2Cre+Leprflox/flox mice (p < 0.05) vs. their respective controls. In conclusion, reconstitution of Lepr in cells of myeloid lineage has beneficial effects on glucose metabolism in male mice.
Asunto(s)
Glucosa/metabolismo , Leptina/metabolismo , Células Mieloides/metabolismo , Transducción de Señal , Animales , Biomarcadores , Glucemia/metabolismo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Metabolismo Energético , Técnicas de Silenciamiento del Gen , Homeostasis , Leptina/genética , Masculino , Enfermedades Metabólicas/etiología , Enfermedades Metabólicas/metabolismo , RatonesRESUMEN
miRNAs have crucial functions in many biological processes and are candidate biomarkers of disease. Here, we show that miR-216a is a conserved, pancreas-specific miRNA with important roles in pancreatic islet and acinar cells. Deletion of miR-216a in mice leads to a reduction in islet size, ß-cell mass, and insulin levels. Single-cell RNA sequencing reveals a subpopulation of ß-cells with upregulated acinar cell markers under a high-fat diet. miR-216a is induced by TGF-ß signaling, and inhibition of miR-216a increases apoptosis and decreases cell proliferation in pancreatic cells. Deletion of miR-216a in the pancreatic cancer-prone mouse line KrasG12D;Ptf1aCreER reduces the propensity of pancreatic cancer precursor lesions. Notably, circulating miR-216a levels are elevated in both mice and humans with pancreatic cancer. Collectively, our study gives insights into how ß-cell mass and acinar cell growth are modulated by a pancreas-specific miRNA and also suggests miR-216a as a potential biomarker for diagnosis of pancreatic diseases.
Asunto(s)
Progresión de la Enfermedad , Eliminación de Gen , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , MicroARNs/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Animales , Apoptosis , Secuencia de Bases , Línea Celular Tumoral , Movimiento Celular , Dieta Alta en Grasa , Humanos , Secreción de Insulina , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/metabolismo , Especificidad de Órganos , RatasRESUMEN
BACKGROUND: Individuals with diabetes are at a greater risk of hospitalization and mortality resulting from viral, bacterial, and fungal infections. The coronavirus disease-2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has spread quickly to more than 213 countries and claimed 395,779 lives as of June 7, 2020. Notably, in several studies, diabetes is one of the most reported comorbidities in patients with severe COVID-19. SCOPE OF REVIEW: In this review, I summarize the clinical data on the risk for infectious diseases in individuals with diabetes while highlighting the mechanisms for altered immune regulation. The focus is on coronaviruses. Based on the new clinical data obtained from COVID-19 patients, a discussion of mechanisms, such as cytokine storm, pulmonary and endothelial dysfunction, and hypercoagulation, that may render individuals with diabetes more vulnerable to COVID-19 is provided. MAJOR CONCLUSIONS: Epidemiological studies show that poorly controlled diabetes is a risk factor for various infectious diseases. Given the global burden of diabetes and the pandemic nature of coronaviruses, understanding how diabetes affects COVID-19 severity is critical to designing tailored treatments and clinical management of individuals affected by diabetes.
Asunto(s)
Betacoronavirus , Infecciones por Coronavirus/epidemiología , Diabetes Mellitus Tipo 1/epidemiología , Diabetes Mellitus Tipo 2/epidemiología , Neumonía Viral/epidemiología , Adulto , Anciano , Animales , COVID-19 , Niño , Comorbilidad , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/virología , Citocinas/metabolismo , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 2/inmunología , Femenino , Humanos , Inmunidad Celular , Inmunidad Innata , Incidencia , Masculino , Ratones , Persona de Mediana Edad , Pandemias , Neumonía Viral/inmunología , Neumonía Viral/patología , Neumonía Viral/virología , Factores de Riesgo , SARS-CoV-2RESUMEN
Hypoxia-inducible factor 1 (HIF-1) is the key transcription factor regulating hypoxia-dependent gene expression. Lack of oxygen stabilizes HIF-1, which in turn modulates the gene expression pattern to adapt cells to the hypoxic environment. Activation of HIF-1 is also detected in most solid tumors and supports tumor growth through the expression of target genes that are involved in processes like cell proliferation, energy metabolism, and oxygen delivery. Poly(ADP-ribose) polymerase 1 (PARP1) is a chromatin-associated protein, which was shown to regulate transcription. Here we report that chronic myelogenous leukemia cells expressing small interfering RNA against PARP1, which were injected into wild-type mice expressing PARP1, showed tumor growth with increased levels of necrosis, limited vascularization, and reduced expression of GLUT-1. Of note, PARP1-deficient cells showed a reduced HIF-1 transcriptional activation that was dependent on PARP1 enzymatic activity. PARP1 neither influenced binding of HIF-1 to its hypoxic response element nor changed HIF-1alpha protein levels in hypoxic cells. However, PARP1 formed a complex with HIF-1alpha through direct protein interaction and coactivated HIF-1alpha-dependent gene expression. These findings provide convincing evidence that wild-type mice expressing PARP1 cannot compensate for the loss of PARP1 in tumor cells and strengthen the importance of the role of PARP1 as a transcriptional coactivator of HIF-1-dependent gene expression during tumor progression.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias/genética , Neoplasias/patología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Animales , Muerte Celular , Proliferación Celular , Supervivencia Celular , Regulación hacia Abajo/genética , Fibroblastos/enzimología , Fibroblastos/metabolismo , Células HeLa , Humanos , Células K562 , Pulmón/citología , Pulmón/enzimología , Ratones , Ratones Desnudos , Necrosis , Neoplasias/irrigación sanguínea , Poli(ADP-Ribosa) Polimerasas/deficiencia , Poli(ADP-Ribosa) Polimerasas/genética , Unión Proteica , ARN Interferente Pequeño , Transducción de Señal , Transcripción Genética , Activación Transcripcional/genéticaRESUMEN
Childhood obesity and early rapid growth increase the risk for type 2 diabetes. Such early overnutrition can be modeled in mice by reducing litter size. We investigated the effects of early overnutrition and increased dietary fat intake on ß cell function in Swiss Webster mice. On a moderate-fat diet, early overnutrition accelerated weight gain and induced hyperinsulinemia in pups. Early overnutrition males exhibited higher ß cell mass but reduced islet insulin content and Pdx1 expression. Males had a high diabetes incidence that was increased by early overnutrition, characterized by a progressive increase in insulin secretion as well as ß cell death, indicated by histological analysis and increased circulating miR-375 levels. Females maintained normoglycemia throughout life. High-fat diet (HFD) increased diabetes incidence in males, whereas low-fat diet was completely protective. This protective effect was abolished in early overnutrition males transiently exposed to HFD in early life. Although Swiss Webster mice are not known to be diabetes-prone, the high diabetes incidence suggests an underlying genetic susceptibility that can be induced by overnutrition and increased dietary fat intake in early life. Thus, the nutritional environment in early life may impact long-term ß cell function and increase diabetes risk, particularly in genetically susceptible individuals.
Asunto(s)
Diabetes Mellitus Experimental/patología , Dieta Alta en Grasa/efectos adversos , Proteínas de Homeodominio/metabolismo , Resistencia a la Insulina , Células Secretoras de Insulina/patología , Hipernutrición/complicaciones , Transactivadores/metabolismo , Animales , Glucemia/metabolismo , Peso Corporal , Diabetes Mellitus Experimental/etiología , Diabetes Mellitus Experimental/metabolismo , Femenino , Proteínas de Homeodominio/genética , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Masculino , Ratones , Transactivadores/genética , Aumento de PesoRESUMEN
Type 1 diabetes (T1D) is an autoimmune disease that is clinically silent until the majority of ß cells are destroyed. There is an unmet need for reliable and cost-effective biomarkers to predict and diagnose diabetes at an early stage. A number of stable microRNAs (miRNAs) have been reported in serum and plasma and are now being investigated as biomarkers of different diseases. We measured the levels of 745 miRNAs in sera of children with recent-onset T1D and age-matched controls using locked nucleic acid-enhanced (LNA-enhanced) quantitative PCR profiling. Thirty-five miRNAs were significantly different between the groups, and 27 miRNAs were elevated in T1D. Good discriminating power was obtained for 6 miRNAs (miR-454-3p, miR-222-3p, miR-144-5p, miR-345-5p, miR-24-3p, and miR-140-5p), which were not elevated at later stages of diabetes. In silico pathway analysis, based on inferred miRNA target genes, associated glycosaminoglycan biosynthesis as well as PI3K/Akt, MAPK, and Wnt signaling pathways with early stages of T1D. Among the 27 upregulated miRNAs in T1D, 2 miRNAs significantly correlated with hemoglobin A1c (HbA1c), as did 5 of 8 downregulated miRNAs. A total of 134 miRNAs significantly correlated with HbA1c when stratifying hyperglycemia-induced miRNAs from T1D-specific miRNAs. In conclusion, we have identified a serum miRNA pattern of recent-onset T1D and signaling pathways that may be involved in its pathogenesis.
Asunto(s)
Diabetes Mellitus Tipo 1/genética , MicroARNs/sangre , Estudios de Casos y Controles , Niño , Simulación por Computador , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/metabolismo , Regulación hacia Abajo , Femenino , Hemoglobina Glucada/metabolismo , Glicosaminoglicanos/biosíntesis , Humanos , Sistema de Señalización de MAP Quinasas , Masculino , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transcriptoma , Regulación hacia Arriba , Vía de Señalización WntRESUMEN
Pluripotent human embryonic stem cells (hESCs) are a potential source of transplantable cells for treating patients with diabetes. To investigate the impact of the host recipient on hESC-derived pancreatic progenitor cell maturation, cells were transplanted into immunodeficient SCID-beige mice or nude rats. Following the transplant, basal human C-peptide levels were consistently higher in mice compared with rats, but only rats showed robust meal- and glucose-responsive human C-peptide secretion by 19-21 weeks. Grafts from rats contained a higher proportion of insulin:glucagon immunoreactivity, fewer exocrine cells, and improved expression of mature ß cell markers compared with mice. Moreover, ECM-related genes were enriched, the collagen network was denser, and blood vessels were more intricately integrated into the engrafted endocrine tissue in rats relative to mice. Overall, hESC-derived pancreatic progenitor cells matured faster in nude rats compared with SCID-beige mice, indicating that the host recipient can greatly influence the fate of immature pancreatic progenitor cells post-transplantation.
Asunto(s)
Células Madre Embrionarias/citología , Células Secretoras de Insulina/citología , Páncreas/citología , Trasplante de Células Madre , Animales , Péptido C/análisis , Diferenciación Celular , Línea Celular , Células Cultivadas , Femenino , Humanos , Masculino , Ratones , Ratones SCID , Persona de Mediana Edad , Ratas , Ratas Desnudas , Especificidad de la EspecieRESUMEN
Human embryonic stem cells (hESCs) were used as a model system of human pancreas development to study characteristics of the polyhormonal cells that arise during fetal pancreas development. HESCs were differentiated into fetal-like pancreatic cells in vitro using a 33-day, 7-stage protocol. Cultures were ~90-95% PDX1-positive by day (d) 11 and 70-75% NKX6.1-positive by d17. Polyhormonal cells were scattered at d17, but developed into islet-like clusters that expressed key transcription factors by d33. Human C-peptide and glucagon secretion were first detected at d17 and increased thereafter in parallel with INS and GCG transcript levels. HESC-derived cells were responsive to KCl and arginine, but not glucose in perifusion studies. Compared to adult human islets, hESC-derived cells expressed ~10-fold higher levels of glucose transporter 1 (GLUT1) mRNA, but similar levels of glucokinase (GCK). In situ hybridization confirmed the presence of GLUT1 transcript within endocrine cells. However, GLUT1 protein was excluded from this population and was instead observed predominantly in non-endocrine cells, whereas GCK was co-expressed in insulin-positive cells. In rubidium efflux assays, hESC-derived cells displayed mild potassium channel activity, but no responsiveness to glucose, metabolic inhibitors or glibenclamide. Western blotting experiments revealed that the higher molecular weight SUR1 band was absent in hESC-derived cells, suggesting a lack of functional KATP channels at the cell surface. In addition, KATP channel subunit transcript levels were not at a 1:1 ratio, as would be expected (SUR1 levels were ~5-fold lower than KIR6.2). Various ratios of SUR1:KIR6.2 plasmids were transfected into COSM6 cells and rubidium efflux was found to be particularly sensitive to a reduction in SUR1. These data suggest that an impaired ratio of SUR1:KIR6.2 may contribute to the observed KATP channel defects in hESC-derived islet endocrine cells, and along with lack of GLUT1, may explain the absence of glucose-stimulated insulin secretion.
Asunto(s)
Células Madre Embrionarias/citología , Células Secretoras de Insulina/metabolismo , Adenosina Trifosfato/metabolismo , Péptido C/metabolismo , Diferenciación Celular , Células Endocrinas/citología , Células Endocrinas/metabolismo , Glucoquinasa/metabolismo , Transportador de Glucosa de Tipo 1/metabolismo , Gliburida/farmacología , Hormonas/metabolismo , Humanos , Hipoglucemiantes/farmacología , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Canales KATP/metabolismo , Canales de Potasio de Rectificación Interna/genética , Canales de Potasio de Rectificación Interna/metabolismo , Receptores de Sulfonilureas/genética , Receptores de Sulfonilureas/metabolismoRESUMEN
Type 1 diabetes is a progressive autoimmune disease that is largely silent in its initial stages. Yet, sensitive methods for detection of ß-cell death and prediction and prevention of diabetes are lacking. Micro-RNAs (miRNAs) have been found at high concentrations in body fluids. Here in this study we sought to determine whether an islet enriched miRNA, miR-375, is a suitable blood marker to detect ß-cell death and predict diabetes in mice. We measured miR-375 levels by quantitative RT-PCR in plasma samples of streptozotocin (STZ)-treated C57BL/6 mice and nonobese diabetic (NOD) mice. We also measured miR-375 levels in media samples of cytokine- or STZ-treated islets in the presence or absence of cell-death inhibitors. High-dose STZ administration dramatically increased circulating miR-375 levels, prior to the onset of hyperglycemia. Similarly, in the NOD mouse model of autoimmune diabetes, circulating miR-375 levels were significantly increased 2 weeks before diabetes onset. Moreover, cytokine- and STZ-induced cell death in isolated mouse islets produced a striking increase in extracellular miR-375 levels, which was reduced by cell death inhibitors. These data suggest that circulating miR-375 can be used as a marker of ß-cell death and potential predictor of diabetes.
Asunto(s)
Biomarcadores/sangre , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/sangre , Células Secretoras de Insulina/patología , MicroARNs/sangre , Animales , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , EstreptozocinaRESUMEN
Poly(ADP-ribose)polymerase-1 (PARP1) is a chromatin-associated enzyme that was described to affect chromatin compaction. Previous reports suggested a dynamic modulation of the chromatin landscape during adipocyte differentiation. We thus hypothesized that PARP1 plays an important transcriptional role in adipogenesis and metabolism and therefore used adipocyte development and function as a model to elucidate the molecular action of PARP1 in obesity-related diseases. Our results show that PARP1-dependent ADP-ribose polymer (PAR) formation increases during adipocyte development and, at late time points of adipogenesis, is involved in the sustained expression of PPARγ2 and of PPARγ2 target genes. During adipogenesis, PARP1 was recruited to PPARγ2 target genes such as CD36 or aP2 in a PAR-dependent manner. Our results also reveal a PAR-dependent decrease in repressory histone marks (e.g. H3K9me3) and an increase in stimulatory marks (e.g. H3K4me3) at the PPARγ2 promoter, suggesting that PARP1 may exert its regulatory function during adipogenesis by altering histone marks. Interestingly, activation of PARP1 enzymatic activity was prevented with a topoisomerase II inhibitor. These data hint at topoisomerase II-dependent, transient, site-specific double-strand DNA breaks as the cause for poly(ADP)-ribose formation, adipogenic gene expression, and adipocyte function. Together, our study identifies PARP1 as a critical regulator of PPARγ2-dependent gene expression with implications in adipocyte function and obesity-related disease models.