RESUMO
Despite >80 years of clinical experience with coagulation factor VIII (FVIII) inhibitors, surprisingly little is known about the in vivo mechanism of this most serious complication of replacement therapy for hemophilia A. These neutralizing antidrug alloantibodies arise in â¼30% of patients. Inhibitor formation is T-cell dependent, but events leading up to helper T-cell activation have been elusive because of, in part, the complex anatomy and cellular makeup of the spleen. Here, we show that FVIII antigen presentation to CD4+ T cells critically depends on a select set of several anatomically distinct antigen-presenting cells, whereby marginal zone B cells and marginal zone and marginal metallophilic macrophages but not red pulp macrophages (RPMFs) participate in shuttling FVIII to the white pulp in which conventional dendritic cells (DCs) prime helper T cells, which then differentiate into follicular helper T (Tfh) cells. Toll-like receptor 9 stimulation accelerated Tfh cell responses and germinal center and inhibitor formation, whereas systemic administration of FVIII alone in hemophilia A mice increased frequencies of monocyte-derived and plasmacytoid DCs. Moreover, FVIII enhanced T-cell proliferation to another protein antigen (ovalbumin), and inflammatory signaling-deficient mice were less likely to develop inhibitors, indicating that FVIII may have intrinsic immunostimulatory properties. Ovalbumin, which, unlike FVIII, is absorbed into the RPMF compartment, fails to elicit T-cell proliferative and antibody responses when administered at the same dose as FVIII. Altogether, we propose that an antigen trafficking pattern that results in efficient in vivo delivery to DCs and inflammatory signaling, shape the immunogenicity of FVIII.
Assuntos
Linfócitos T CD4-Positivos , Fator VIII , Hemofilia A , Hemostáticos , Animais , Camundongos , Células Dendríticas/metabolismo , Fator VIII/imunologia , Fator VIII/uso terapêutico , Hemofilia A/tratamento farmacológico , Hemostáticos/imunologia , Hemostáticos/uso terapêutico , Ovalbumina/imunologiaRESUMO
AIMS/HYPOTHESIS: Pancreatic beta cells are subjected to exogenous damaging factors such as proinflammatory cytokines or excess glucose that can cause accumulation of damage-inducing reactive oxygen species during the pathogenesis of diabetes. We and others have shown that beta cell autophagy can reduce reactive oxygen species to protect against apoptosis. While impaired islet autophagy has been demonstrated in human type 2 diabetes, it is unknown if islet autophagy is perturbed in the pathogenesis of type 1 diabetes. We hypothesised that beta cell autophagy is dysfunctional in type 1 diabetes, and that there is a progressive loss during early diabetes development. METHODS: Pancreases were collected from chloroquine-injected and non-injected non-obese diabetes-resistant (NOR) and non-obese diabetic (NOD) mice. Age- and BMI-matched pancreas tissue sections from human organ donors (N = 34) were obtained from the Network for Pancreatic Organ Donors with Diabetes (nPOD). Tissue sections were stained with antibodies against proinsulin or insulin (beta cell markers), microtubule-associated protein 1 light chain 3 A/B (LC3A/B; autophagosome marker), lysosomal-associated membrane protein 1 (LAMP1; lysosome marker) and p62 (autophagy adaptor). Images collected on a scanning laser confocal microscope were analysed with CellProfiler and ImageJ. Secondary lysosomes and telolysosomes were assessed in electron micrographs of human pancreatic tissue sections (n = 12), and energy dispersive x-ray analysis was performed to assess distribution of elements (n = 5). RESULTS: We observed increased autophagosome numbers in islets of diabetic NOD mice (p = 0.008) and increased p62 in islets of both non-diabetic and diabetic NOD mice (p < 0.001) vs NOR mice. There was also a reduction in LC3-LAMP1 colocalisation in islets of diabetic NOD mice compared with both non-diabetic NOD (p < 0.001) and NOR mice (p < 0.001). Chloroquine elicited accumulation of autophagosomes in the islets of NOR (p = 0.003) and non-diabetic NOD mice (p < 0.001), but not in islets of diabetic NOD mice; and stimulated accumulation of p62 in NOR (p < 0.001), but not in NOD mice. We observed reduced LC3-LAMP1 colocalisation (p < 0.001) in residual beta cells of human donors with type 1 diabetes vs non-diabetic participants. We also observed reduced colocalisation of proinsulin with LAMP1 in donors with type 1 diabetes (p < 0.001). Electron microscopy also revealed accumulation of telolysosomes with nitrogen-dense rings in beta cells of autoantibody-positive donors (p = 0.002). CONCLUSIONS/INTERPRETATION: We provide evidence of islet macroautophagy/crinophagy impairment in human type 1 diabetes. We also document accumulation of telolysosomes with peripheral nitrogen in beta cells of autoantibody-positive donors, demonstrating altered lysosome content that may be associated with lysosome dysfunction before clinical hyperglycaemia. Similar macroautophagy impairments are present in the NOD mouse model of type 1 diabetes.
Assuntos
Diabetes Mellitus Tipo 1/patologia , Células Secretoras de Insulina/patologia , Lisossomos/patologia , Macroautofagia , Adolescente , Adulto , Animais , Proteínas Relacionadas à Autofagia/metabolismo , Estudos de Casos e Controles , Diabetes Mellitus Tipo 1/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Células Secretoras de Insulina/metabolismo , Lisossomos/metabolismo , Masculino , Camundongos Endogâmicos NOD , Transdução de Sinais , Adulto JovemRESUMO
Type 1 diabetes is an insulin-dependent, autoimmune disease where the pancreatic ß cells are destroyed resulting in hyperglycemia. This multifactorial disease involves multiple environmental and genetic factors, and has no clear etiology. Accumulating evidence suggests that early signaling defects within the ß cells may promote a change in the local immune milieu leading to autoimmunity. Therefore, many studies have been focused on intrinsic ß-cell mechanisms that aid in the restoration of cellular homeostasis under environmental conditions that cause dysfunction. One of these intrinsic mechanisms to promote homeostasis is autophagy, defects which are clearly linked with ß-cell dysfunction in the context of type 2 diabetes. Recent studies have now also pointed towards ß-cell autophagy defects in the context of type 1 diabetes. In this perspectives review, we will discuss the evidence supporting a role for ß-cell autophagy in the pathogenesis of type 1 diabetes, including a potential role for unconventional secretion of autophagosomes/lysosomes in the changing dialogue between the ß cell and immune cells.
Assuntos
Autofagia , Diabetes Mellitus Tipo 1/fisiopatologia , Células Secretoras de Insulina/fisiologia , Animais , Humanos , Lisossomos/fisiologiaRESUMO
In type 1 diabetes, an autoimmune event increases oxidative stress in islet ß cells, giving rise to cellular dysfunction and apoptosis. Lipoxygenases are enzymes that catalyze the oxygenation of polyunsaturated fatty acids that can form lipid metabolites involved in several biological functions, including oxidative stress. 12-Lipoxygenase and 12/15-lipoxygenase are related but distinct enzymes that are expressed in pancreatic islets, but their relative contributions to oxidative stress in these regions are still being elucidated. In this study, we used mice with global genetic deletion of the genes encoding 12-lipoxygenase (arachidonate 12-lipoxygenase, 12S type [Alox12]) or 12/15-lipoxygenase (Alox15) to compare the influence of each gene deletion on ß cell function and survival in response to the ß cell toxin streptozotocin. Alox12-/- mice exhibited greater impairment in glucose tolerance following streptozotocin exposure than WT mice, whereas Alox15-/- mice were protected against dysglycemia. These changes were accompanied by evidence of islet oxidative stress in Alox12-/- mice and reduced oxidative stress in Alox15-/- mice, consistent with alterations in the expression of the antioxidant response enzymes in islets from these mice. Additionally, islets from Alox12-/- mice displayed a compensatory increase in Alox15 gene expression, and treatment of these mice with the 12/15-lipoxygenase inhibitor ML-351 rescued the dysglycemic phenotype. Collectively, these results indicate that Alox12 loss activates a compensatory increase in Alox15 that sensitizes mouse ß cells to oxidative stress.
Assuntos
Araquidonato 12-Lipoxigenase/deficiência , Araquidonato 15-Lipoxigenase/biossíntese , Regulação Enzimológica da Expressão Gênica , Células Secretoras de Insulina/enzimologia , Estresse Oxidativo , Animais , Araquidonato 12-Lipoxigenase/biossíntese , Araquidonato 12-Lipoxigenase/genética , Araquidonato 15-Lipoxigenase/genética , Deleção de Genes , Isoxazóis/farmacologia , Camundongos , Camundongos Knockout , Naftalenos/farmacologia , Estreptozocina/toxicidadeRESUMO
IL-6 is a pleiotropic cytokine with complex roles in inflammation and metabolic disease. The role of IL-6 as a pro- or anti-inflammatory cytokine is still unclear. Within the pancreatic islet, IL-6 stimulates secretion of the prosurvival incretin hormone glucagon-like peptide 1 (GLP-1) by α cells and acts directly on ß cells to stimulate insulin secretion in vitro Uncovering physiologic mechanisms promoting ß-cell survival under conditions of inflammation and stress can identify important pathways for diabetes prevention and treatment. Given the established role of GLP-1 in promoting ß-cell survival, we hypothesized that IL-6 may also directly protect ß cells from apoptosis. Herein, we show that IL-6 robustly activates signal transducer and activator of transcription 3 (STAT3), a transcription factor that is involved in autophagy. IL-6 stimulates LC3 conversion and autophagosome formation in cultured ß cells. In vivo IL-6 infusion stimulates a robust increase in lysosomes in the pancreas that is restricted to the islet. Autophagy is critical for ß-cell homeostasis, particularly under conditions of stress and increased insulin demand. The stimulation of autophagy by IL-6 is regulated via multiple complementary mechanisms including inhibition of mammalian target of rapamycin complex 1 (mTORC1) and activation of Akt, ultimately leading to increases in autophagy enzyme production. Pretreatment with IL-6 renders ß cells resistant to apoptosis induced by proinflammatory cytokines, and inhibition of autophagy with chloroquine prevents the ability of IL-6 to protect from apoptosis. Importantly, we find that IL-6 can activate STAT3 and the autophagy enzyme GABARAPL1 in human islets. We also see evidence of decreased IL-6 pathway signaling in islets from donors with type 2 diabetes. On the basis of our results, we propose direct stimulation of autophagy as a novel mechanism for IL-6-mediated protection of ß cells from stress-induced apoptosis.-Linnemann, A. K., Blumer, J., Marasco, M. R., Battiola, T. J., Umhoefer, H. M., Han, J. Y., Lamming, D. W., Davis, D. B. Interleukin 6 protects pancreatic ß cells from apoptosis by stimulation of autophagy.
Assuntos
Apoptose/fisiologia , Autofagia/fisiologia , Células Secretoras de Insulina/metabolismo , Interleucina-6/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular , Diabetes Mellitus Tipo 2 , Regulação da Expressão Gênica , Peptídeo 1 Semelhante ao Glucagon/genética , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Humanos , Bombas de Infusão Implantáveis , Interleucina-6/genética , Interleucina-6/farmacologia , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Ratos , Proteínas Recombinantes , Transdução de SinaisRESUMO
GATA factors interact with simple DNA motifs (WGATAR) to regulate critical processes, including hematopoiesis, but very few WGATAR motifs are occupied in genomes. Given the rudimentary knowledge of mechanisms underlying this restriction and how GATA factors establish genetic networks, we used ChIP-seq to define GATA-1 and GATA-2 occupancy genome-wide in erythroid cells. Coupled with genetic complementation analysis and transcriptional profiling, these studies revealed a rich collection of targets containing a characteristic binding motif of greater complexity than WGATAR. GATA factors occupied loci encoding multiple components of the Scl/TAL1 complex, a master regulator of hematopoiesis and leukemogenic target. Mechanistic analyses provided evidence for crossregulatory and autoregulatory interactions among components of this complex, including GATA-2 induction of the hematopoietic corepressor ETO-2 and an ETO-2-negative autoregulatory loop. These results establish fundamental principles underlying GATA factor mechanisms in chromatin and illustrate a complex network of considerable importance for the control of hematopoiesis.
Assuntos
Cromatina/metabolismo , Fator de Transcrição GATA1/metabolismo , Fator de Transcrição GATA2/metabolismo , Genoma Humano/genética , Sistema Hematopoético/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Imunoprecipitação da Cromatina , Biologia Computacional , Perfilação da Expressão Gênica , Loci Gênicos , Homeostase , Humanos , Células K562 , Leucemia/metabolismo , Leucemia/patologia , Camundongos , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Análise de Sequência de DNA , Proteína 1 de Leucemia Linfocítica Aguda de Células TRESUMO
Vascular endothelial dysfunction underlies the genesis and progression of numerous diseases. Although the GATA transcription factor GATA-2 is expressed in endothelial cells and is implicated in coronary heart disease, it has been studied predominantly as a master regulator of hematopoiesis. Because many questions regarding GATA-2 function in the vascular biology realm remain unanswered, we used ChIP sequencing and loss-of-function strategies to define the GATA-2-instigated genetic network in human endothelial cells. In contrast to erythroid cells, GATA-2 occupied a unique target gene ensemble consisting of genes encoding key determinants of endothelial cell identity and inflammation. GATA-2-occupied sites characteristically contained motifs that bind activator protein-1 (AP-1), a pivotal regulator of inflammatory genes. GATA-2 frequently occupied the same chromatin sites as c-JUN and c-FOS, heterodimeric components of AP-1. Although all three components were required for maximal AP-1 target gene expression, GATA-2 was not required for AP-1 chromatin occupancy. GATA-2 conferred maximal phosphorylation of chromatin-bound c-JUN at Ser-73, which stimulates AP-1-dependent transactivation, in a chromosomal context-dependent manner. This work establishes a link between a GATA factor and inflammatory genes, mechanistic insights underlying GATA-2-AP-1 cooperativity and a rigorous genetic framework for understanding GATA-2 function in normal and pathophysiological vascular states.
Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Fator de Transcrição GATA2/genética , Redes Reguladoras de Genes , Fator de Transcrição AP-1/genética , Cromatina/metabolismo , Células Endoteliais/fisiologia , Células Eritroides , Genes fos , Genes jun , Humanos , Inflamação/genéticaRESUMO
Recently, a novel type 1 diabetes association locus was identified at human chromosome 6p31.3, and transcription factor 19 (TCF19) is a likely causal gene. Little is known about Tcf19, and we now show that it plays a role in both proliferation and apoptosis in insulinoma cells. Tcf19 is expressed in mouse and human islets, with increasing mRNA expression in nondiabetic obesity. The expression of Tcf19 is correlated with ß-cell mass expansion, suggesting that it may be a transcriptional regulator of ß-cell mass. Increasing proliferation and decreasing apoptotic cell death are two strategies to increase pancreatic ß-cell mass and prevent or delay diabetes. siRNA-mediated knockdown of Tcf19 in the INS-1 insulinoma cell line, a ß-cell model, results in a decrease in proliferation and an increase in apoptosis. There was a significant reduction in the expression of numerous cell cycle genes from the late G1 phase through the M phase, and cells were arrested at the G1/S checkpoint. We also observed increased apoptosis and susceptibility to endoplasmic reticulum (ER) stress after Tcf19 knockdown. There was a reduction in expression of genes important for the maintenance of ER homeostasis (Bip, p58(IPK), Edem1, and calreticulin) and an increase in proapoptotic genes (Bim, Bid, Nix, Gadd34, and Pdia2). Therefore, Tcf19 is necessary for both proliferation and survival and is a novel regulator of these pathways.
Assuntos
Ciclo Celular/fisiologia , Diabetes Mellitus/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Células Secretoras de Insulina/metabolismo , Fatores de Transcrição/metabolismo , Animais , Ciclo Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Diabetes Mellitus/genética , Diabetes Mellitus/patologia , Humanos , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA/química , RNA/genética , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: T1D is an autoimmune disease in which pancreatic islets of Langerhans are infiltrated by immune cells resulting in the specific destruction of insulin-producing islet beta cells. Our understanding of the factors leading to islet infiltration and the interplay of the immune cells with target beta cells is incomplete, especially in human disease. While murine models of T1D have provided crucial information for both beta cell and autoimmune cell function, the translation of successful therapies in the murine model to human disease has been a challenge. SCOPE OF REVIEW: Here, we discuss current state of the art and consider knowledge gaps concerning the interface of the islet beta cell with immune infiltrates, with a focus on T cells. We discuss pancreatic and immune cell phenotypes and their impact on cell function in health and disease, which we deem important to investigate further to attain a more comprehensive understanding of human T1D disease etiology. MAJOR CONCLUSIONS: The last years have seen accelerated development of approaches that allow comprehensive study of human T1D. Critically, recent studies have contributed to our revised understanding that the pancreatic beta cell assumes an active role, rather than a passive position, during autoimmune disease progression. The T cell-beta cell interface is a critical axis that dictates beta cell fate and shapes autoimmune responses. This includes the state of the beta cell after processing internal and external cues (e.g., stress, inflammation, genetic risk) that that contributes to the breaking of tolerance by hyperexpression of human leukocyte antigen (HLA) class I with presentation of native and neoepitopes and secretion of chemotactic factors to attract immune cells. We anticipate that emerging insights about the molecular and cellular aspects of disease initiation and progression processes will catalyze the development of novel and innovative intervention points to provide additional therapies to individuals affected by T1D.
Assuntos
Diabetes Mellitus Tipo 1 , Células Secretoras de Insulina , Ilhotas Pancreáticas , Humanos , Camundongos , Animais , Diabetes Mellitus Tipo 1/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Pâncreas/metabolismo , Fatores de RiscoRESUMO
Human islet transplantations into rodent models are an essential tool to aid in the development and testing of islet and cellular-based therapies for diabetes prevention and treatment. Through the ability to evaluate human islets in an in vivo setting, these studies allow for experimental approaches to answer questions surrounding normal and disease pathophysiology that cannot be answered using other in vitro and in vivo techniques alone. Intravital microscopy enables imaging of tissues in living organisms with dynamic temporal resolution and can be employed to measure biological processes in transplanted human islets revealing how experimental variables can influence engraftment, and transplant survival and function. A key consideration in experimental design for transplant imaging is the surgical placement site, which is guided by the presence of vasculature to aid in functional engraftment of the islets and promote their survival. Here, we review transplantation sites and mouse models used to study beta cell biology in vivo using intravital microscopy and we highlight fundamental observations made possible using this methodology.
Assuntos
Células Secretoras de Insulina , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Camundongos , Animais , Humanos , Transplante das Ilhotas Pancreáticas/métodos , Ilhotas Pancreáticas/diagnóstico por imagem , Ilhotas Pancreáticas/irrigação sanguínea , Sobrevivência de Enxerto , Microscopia Intravital , Modelos Animais de DoençasRESUMO
Metabolic dysfunction of ß-cells has been implicated as a contributor to diabetes pathogenesis, and efforts are ongoing to optimize analytical techniques that evaluate islet metabolism. High-resolution respirometry offers sensitive measurements of the respiratory effects of metabolic substrates and customizable manipulation of electron transport chain components, though the delicate nature of islets can pose challenges to conventional analyses. An affordable and reliable option for respirometry is the Oroboros Oxygraph-2 K system, which utilizes a stir bar to circulate reagents around cells. While this technique may be suitable for individual cells or mitochondria, the continual force exerted by the stir bar can have damaging effects on islet integrity. Herein, we demonstrate the protective benefits of a novel 3D-printed islet stabilization device and highlight the destructive effects of conventional Oxygraph analysis on islet integrity. Islet containment did not inhibit cellular responses to metabolic modulatory drugs, as indicated by robust fluctuations in oxygen consumption rates. The average size of wild-type mouse islets was significantly reduced following a standard Mito Stress Test within Oxygraph chambers, with a clear disruption in islet morphology and viability. Alternatively, containment of the islets within the interior chamber of the islet stabilization device yielded preservation of both islet morphology and increased cell viability/survival after respirometry analysis. Collectively, our study introduces a new and easily accessible tool to improve conventional Oxygraph respirometry of pancreatic islets by preserving natural islet structure and function throughout metabolic analysis.
Assuntos
Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Animais , Sobrevivência Celular , Ilhotas Pancreáticas/metabolismo , Camundongos , Mitocôndrias/metabolismo , Consumo de OxigênioRESUMO
The glucagon-like peptide-1 receptor (GLP1R) is expressed in peripheral tissues and the brain, where it exerts pleiotropic actions on metabolic and inflammatory processes. Detection and visualization of GLP1R remains challenging, partly due to a lack of validated reagents. Previously, we generated LUXendins, antagonistic red and far-red fluorescent probes for specific labeling of GLP1R in live and fixed cells/tissues. We now extend this concept to the green and near-infrared color ranges by synthesizing and testing LUXendin492, LUXendin551, LUXendin615, and LUXendin762. All four probes brightly and specifically label GLP1R in cells and pancreatic islets. Further, LUXendin551 acts as a chemical beta cell reporter in preclinical rodent models, while LUXendin762 allows noninvasive imaging, highlighting differentially accessible GLP1R populations. We thus expand the color palette of LUXendins to seven different spectra, opening up a range of experiments using wide-field microscopy available in most labs through super-resolution imaging and whole animal imaging. With this, we expect that LUXendins will continue to generate novel and specific insights into GLP1R biology.
RESUMO
Asthma is a chronic inflammatory lung disease with intermittent flares predominately mediated through memory T cells. Yet, the identity of long-term memory cells that mediate allergic recall responses is not well defined. In this report, using a mouse model of chronic allergen exposure followed by an allergen-free rest period, we characterized a subpopulation of CD4+ T cells that secreted IL-9 as an obligate effector cytokine. IL-9-secreting cells had a resident memory T cell phenotype, and blocking IL-9 during a recall challenge or deleting IL-9 from T cells significantly diminished airway inflammation and airway hyperreactivity. T cells secreted IL-9 in an allergen recall-specific manner, and secretion was amplified by IL-33. Using scRNA-seq and scATAC-seq, we defined the cellular identity of a distinct population of T cells with a proallergic cytokine pattern. Thus, in a recall model of allergic airway inflammation, IL-9 secretion from a multicytokine-producing CD4+ T cell population was required for an allergen recall response.
Assuntos
Asma , Hipersensibilidade , Alérgenos , Linfócitos T CD4-Positivos , Citocinas , Humanos , Inflamação , Interleucina-9RESUMO
Mitochondrial glucose metabolism is essential for stimulated insulin release from pancreatic ß-cells. Whether mitofusin gene expression, and hence, mitochondrial network integrity, is important for glucose or incretin signaling has not previously been explored. Here, we generated mice with ß-cell-selective, adult-restricted deletion knock-out (dKO) of the mitofusin genes Mfn1 and Mfn2 (ßMfn1/2 dKO). ßMfn1/2-dKO mice displayed elevated fed and fasted glycemia and a more than fivefold decrease in plasma insulin. Mitochondrial length, glucose-induced polarization, ATP synthesis, and cytosolic and mitochondrial Ca2+ increases were all reduced in dKO islets. In contrast, oral glucose tolerance was more modestly affected in ßMfn1/2-dKO mice, and glucagon-like peptide 1 or glucose-dependent insulinotropic peptide receptor agonists largely corrected defective glucose-stimulated insulin secretion through enhanced EPAC-dependent signaling. Correspondingly, cAMP increases in the cytosol, as measured with an Epac-camps-based sensor, were exaggerated in dKO mice. Mitochondrial fusion and fission cycles are thus essential in the ß-cell to maintain normal glucose, but not incretin, sensing. These findings broaden our understanding of the roles of mitofusins in ß-cells, the potential contributions of altered mitochondrial dynamics to diabetes development, and the impact of incretins on this process.
Assuntos
GTP Fosfo-Hidrolases , Glucose , Incretinas , Células Secretoras de Insulina , Animais , GTP Fosfo-Hidrolases/genética , Glucose/metabolismo , Glucose/farmacologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Incretinas/metabolismo , Incretinas/farmacologia , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Camundongos , Camundongos KnockoutRESUMO
It is well established that nuclear architecture plays a key role in poising regions of the genome for transcription. This may be achieved using scaffold/matrix attachment regions (S/MARs) that establish loop domains. However, the relationship between changes in the physical structure of the genome as mediated by attachment to the nuclear scaffold/matrix and gene expression is not clearly understood. To define the role of S/MARs in organizing our genome and to resolve the often contradictory loci-specific studies, we have surveyed the S/MARs in HeLa S3 cells on human chromosomes 14-18 by array comparative genomic hybridization. Comparison of LIS (lithium 3,5-diiodosalicylate) extraction to identify SARs and 2 m NaCl extraction to identify MARs revealed that approximately one-half of the sites were in common. The results presented in this study suggest that SARs 5' of a gene are associated with transcript presence whereas MARs contained within a gene are associated with silenced genes. The varied functions of the S/MARs as revealed by the different extraction methods highlights their unique functional contribution.
Assuntos
Expressão Gênica , Regiões de Interação com a Matriz , Matriz Nuclear/genética , Mapeamento Cromossômico , Cromossomos Humanos/química , Cromossomos Humanos/genética , Cromossomos Humanos/metabolismo , Células HeLa , Humanos , Matriz Nuclear/química , Matriz Nuclear/metabolismo , Ligação ProteicaRESUMO
Within the sperm nucleus, the paternal genome remains functionally inert and protected following protamination. This is marked by a structural morphogenesis that is heralded by a striking reduction in nuclear volume. Despite these changes, both human and mouse spermatozoa maintain low levels of nucleosomes that appear non-randomly distributed throughout the genome. These regions may be necessary for organizing higher order genomic structure through interactions with the nuclear matrix. The promoters of this transcriptionally quiescent genome are differentially marked by modified histones that may poise downstream epigenetic effects. This notion is supported by increasing evidence that the embryo inherits these differing levels of chromatin organization. In concert with the suite of RNAs retained in the mature sperm, they may synergistically interact to direct early embryonic gene expression. Irrespective, these features reflect the transcriptional history of spermatogenic differentiation. As such, they may soon be utilized as clinical markers of male fertility. In this review, we explore and discuss how this may be orchestrated.
Assuntos
Núcleo Celular/metabolismo , Cromatina/metabolismo , Matriz Nuclear/metabolismo , RNA/metabolismo , Espermatozoides/metabolismo , Animais , Montagem e Desmontagem da Cromatina , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Masculino , Camundongos , Nucleossomos/metabolismoRESUMO
DNA loop organization by nuclear scaffold/matrix attachment is a key regulator of gene expression that may provide a means to modulate phenotype. We have previously shown that attachment of genes to the NaCl-isolated nuclear matrix correlates with their silencing in HeLa cells. In contrast, expressed genes were associated with the lithium 3,5-diiodosalicylate (LIS)-isolated nuclear scaffold. To define their role in determining phenotype matrix attached regions (MARs) on human chromosomes 14-18 were identified as a function of expression in a primary cell line. The locations of MARs in aortic adventitial fibroblast (AoAF) cells were very stable (r = 0.909) and 96% of genes attached at MARs are silent (P < 0.001). Approximately one-third of the genes uniquely expressed in AoAF cells were associated with the HeLa cell nuclear matrix and silenced. Comparatively, 81% were associated with the AoAF cell nuclear scaffold (P < 0.001) and expressed. This suggests that nuclear scaffold/matrix association mediates a portion of cell type-specific gene expression thereby modulating phenotype. Interestingly, nuclear matrix attachment and thus silencing of specific genes that regulate proliferation and maintain the integrity of the HeLa cell genome suggests that transformation may at least in part be achieved through aberrant nuclear matrix attachment.
Assuntos
Proliferação de Células , Inativação Gênica , Regiões de Interação com a Matriz , Matriz Nuclear/metabolismo , Linhagem Celular , Genoma Humano , Células HeLa , Humanos , Matriz Nuclear/química , Transcrição GênicaRESUMO
Impaired fibrinolysis has long been considered as a risk factor for venous thromboembolism. Fibrin clots formed at physiological concentrations are promising substrates for monitoring fibrinolytic performance as they offer clot microstructures resembling in vivo. Here we introduce a fluorescently labeled fibrin clot lysis assay which leverages a unique annular clot geometry assayed using a microplate reader. A physiologically relevant fibrin clotting formulation was explored to achieve high assay sensitivity while minimizing labeling impact as fluorescence isothiocyanate (FITC)-fibrin(ogen) conjugations significantly affect both fibrin polymerization and fibrinolysis. Clot characteristics were examined using thromboelastography (TEG), turbidity, scanning electron microscopy, and confocal microscopy. Sample fibrinolytic activities at varying plasmin, plasminogen, and tissue plasminogen activator (tPA) concentrations were assessed in the present study and results were compared to an S2251 chromogenic assay. The optimized physiologically relevant clot substrate showed minimal reporter-conjugation impact with nearly physiological clot properties. The assay demonstrated good reproducibility, wide working range, kinetic read ability, low limit of detection, and the capability to distinguish fibrin binding-related lytic performance. In combination with its ease for multiplexing, it also has applications as a convenient platform for assessing patient fibrinolytic potential and screening thrombolytic drug activities in personalized medical applications.
Assuntos
Fibrina/química , Fluoresceína-5-Isotiocianato/química , Trombose/diagnóstico por imagem , Cristalização , Fibrinogênio/química , Microscopia Eletrônica de Varredura , Imagem ÓpticaRESUMO
Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, with an estimated global prevalence of 1 in 4 individuals. Aberrant transcriptional control of gene expression is central to the pathophysiology of metabolic diseases. However, the molecular mechanisms leading to gene dysregulation are not well understood. Histone modifications play important roles in the control of transcription. Acetylation of histone 3 at lysine 9 (H3K9ac) is associated with transcriptional activity and is implicated in transcript elongation by controlling RNA polymerase II (RNAPII) pause-release. Hence, changes in this histone modification may shed information on novel pathways linking transcription control and metabolic dysfunction. Here, we carried out genome-wide analysis of H3K9ac in the liver of mice fed a control or a high-fat diet (an animal model of NAFLD), and asked whether this histone mark associates with changes in gene expression. We found that over 70% of RNAPII peaks in promoter-proximal regions overlapped with H3K9ac, consistent with a role of H3K9ac in the regulation of transcription. When comparing high-fat with control diet, approximately 17% of the differentially expressed genes were associated with changes in H3K9ac in their promoters, showing a strong correlation between changes in H3K9ac signal and gene expression. Overall, our data indicate that in response to a high-fat diet, dysregulated gene expression of a subset of genes may be attributable to changes in transcription elongation driven by H3K9ac. Our results point at an added mechanism of gene regulation that may be important in the development of metabolic diseases.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Regiões Promotoras Genéticas , Elongação da Transcrição Genética/efeitos dos fármacos , Acetilação/efeitos dos fármacos , Animais , Histonas/genética , Masculino , Camundongos , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologiaRESUMO
The spatial architecture of the islets of Langerhans is hypothesized to facilitate synchronized insulin secretion among ß cells, yet testing this in vivo in the intact pancreas is challenging. Robo ßKO mice, in which the genes Robo1 and Robo2 are deleted selectively in ß cells, provide a unique model of altered islet spatial architecture without loss of ß cell differentiation or islet damage from diabetes. Combining Robo ßKO mice with intravital microscopy, we show here that Robo ßKO islets have reduced synchronized intra-islet Ca2+ oscillations among ß cells in vivo. We provide evidence that this loss is not due to a ß cell-intrinsic function of Robo, mis-expression or mis-localization of Cx36 gap junctions, or changes in islet vascularization or innervation, suggesting that the islet architecture itself is required for synchronized Ca2+ oscillations. These results have implications for understanding structure-function relationships in the islets during progression to diabetes as well as engineering islets from stem cells.