RESUMEN
The 2011 discovery of the first rare earth-dependent enzyme in methylotrophic Methylobacterium extorquens AM1 prompted intensive research toward understanding the unique chemistry at play in these systems. This enzyme, an alcohol dehydrogenase (ADH), features a La3+ ion closely associated with redox-active coenzyme pyrroloquinoline quinone (PQQ) and is structurally homologous to the Ca2+-dependent ADH from the same organism. AM1 also produces a periplasmic PQQ-binding protein, PqqT, which we have now structurally characterized to 1.46-Å resolution by X-ray diffraction. This crystal structure reveals a Lys residue hydrogen-bonded to PQQ at the site analogously occupied by a Lewis acidic cation in ADH. Accordingly, we prepared K142A- and K142D-PqqT variants to assess the relevance of this site toward metal binding. Isothermal titration calorimetry experiments and titrations monitored by UV-Vis absorption and emission spectroscopies support that K142D-PqqT binds tightly (Kd = 0.6 ± 0.2 µM) to La3+ in the presence of bound PQQ and produces spectral signatures consistent with those of ADH enzymes. These spectral signatures are not observed for WT- or K142A-variants or upon addition of Ca2+ to PQQ ⸦ K142D-PqqT. Addition of benzyl alcohol to La3+-bound PQQ ⸦ K142D-PqqT (but not Ca2+-bound PQQ ⸦ K142D-PqqT, or La3+-bound PQQ ⸦ WT-PqqT) produces spectroscopic changes associated with PQQ reduction, and chemical trapping experiments reveal the production of benzaldehyde, supporting ADH activity. By creating a metal binding site that mimics native ADH enzymes, we present a rare earth-dependent artificial metalloenzyme primed for future mechanistic, biocatalytic, and biosensing applications.
Asunto(s)
Methylobacterium extorquens , Methylobacterium extorquens/enzimología , Methylobacterium extorquens/metabolismo , Metaloproteínas/química , Metaloproteínas/metabolismo , Alcohol Deshidrogenasa/metabolismo , Alcohol Deshidrogenasa/química , Cristalografía por Rayos X , Cofactor PQQ/metabolismo , Cofactor PQQ/química , Materiales Biomiméticos/química , Materiales Biomiméticos/metabolismo , Metales de Tierras Raras/química , Metales de Tierras Raras/metabolismo , Modelos Moleculares , Lantano/química , Lantano/metabolismoRESUMEN
The life cycle of endogenous retroviruses (ERVs), also called long terminal repeat (LTR) retrotransposons, begins with transcription by RNA polymerase II followed by reverse transcription and re-integration into the host genome. While most ERVs are relics of ancient integration events, "young" proviruses competent for retrotransposition-found in many mammals, but not humans-represent an ongoing threat to host fitness. As a consequence, several restriction pathways have evolved to suppress their activity at both transcriptional and post-transcriptional stages of the viral life cycle. Nevertheless, accumulating evidence has revealed that LTR sequences derived from distantly related ERVs have been exapted as regulatory sequences for many host genes in a wide range of cell types throughout mammalian evolution. Here, we focus on emerging themes from recent studies cataloging the diversity of ERV LTRs acting as important transcriptional regulatory elements in mammals and explore the molecular features that likely account for LTR exaptation in developmental and tissue-specific gene regulation.
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ADN Viral/genética , Retrovirus Endógenos/genética , Regulación Viral de la Expresión Génica , Secuencias Repetidas Terminales , Transcripción Genética , Replicación Viral , Animales , Ensamble y Desensamble de Cromatina , Replicación del ADN , ADN Viral/biosíntesis , ADN Viral/química , Retrovirus Endógenos/crecimiento & desarrollo , Retrovirus Endógenos/metabolismo , Interacciones Huésped-Patógeno , Humanos , Regiones Promotoras Genéticas , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN Largo no Codificante/biosíntesis , ARN Largo no Codificante/genética , ADN Polimerasa Dirigida por ARN/metabolismoRESUMEN
Type 1 diabetes results from the poorly understood process of islet autoimmunity, which ultimately leads to the loss of functional pancreatic beta cells. Mounting evidence supports the notion that the activation and evolution of islet autoimmunity in genetically susceptible people is contingent upon early life exposures affecting the islets, especially beta cells. Here, we review some of the recent advances and studies that highlight the roles of these changes as well as antigen presentation and stress response pathways in beta cells in the onset and propagation of the autoimmune process in type 1 diabetes. Future progress in this area holds promise for advancing islet- and beta cell-directed therapies that could be implemented in the early stages of the disease and could be combined with immunotherapies.
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Diabetes Mellitus Tipo 1 , Células Secretoras de Insulina , Islotes Pancreáticos , Humanos , Diabetes Mellitus Tipo 1/metabolismo , Células Secretoras de Insulina/metabolismo , Autoinmunidad/fisiología , Islotes Pancreáticos/metabolismo , Predisposición Genética a la EnfermedadRESUMEN
Many naturally occurring metalloenzymes are gated by rate-limiting conformational changes, and there exists a critical interplay between macroscopic structural rearrangements of the protein and subatomic changes affecting the electronic structure of embedded metallocofactors. Despite this connection, most artificial metalloproteins (ArMs) are prepared in structurally rigid protein hosts. To better model the natural mechanisms of metalloprotein reactivity, we have developed conformationally switchable ArMs (swArMs) that undergo a large-scale structural rearrangement upon allosteric effector binding. The swArMs reported here contain a Co(dmgH)2(X) cofactor (dmgH = dimethylglyoxime and X = N3-, H3C-, and iPr-). We used UV-vis absorbance and energy-dispersive X-ray fluorescence spectroscopies, along with protein assays, and mass spectrometry to show that these metallocofactors are installed site-specifically and stoichiometrically via direct Co-S cysteine ligation within the Escherichia coli glutamine binding protein (GlnBP). Structural characterization by single-crystal X-ray diffraction unveils the precise positioning and microenvironment of the metallocofactor within the protein fold. Fluorescence, circular dichroism, and infrared spectroscopies, along with isothermal titration calorimetry, reveal that allosteric Gln binding drives a large-scale protein conformational change. In swArMs containing a Co(dmgH)2(CH3) cofactor, we show that the protein stabilizes the otherwise labile Co-S bond relative to the free complex. Kinetics studies performed as a function of temperature and pH reveal that the protein conformational change accelerates this bond dissociation in a pH-dependent fashion. We present swArMs as a robust platform for investigating the interplay between allostery and metallocofactor regulation.
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Metaloproteínas , Metaloproteínas/química , Cristalografía por Rayos X , Escherichia coli/metabolismo , Dicroismo Circular , CinéticaRESUMEN
Women with underlying cardiac conditions have an increased risk of adverse pregnancy outcomes. Counselling reproductive age women with heart disease is important to assist them in deciding whether to pursue pregnancy, to ensure their best cardiovascular status prior to pregnancy, and that they understand the risks of pregnancy for them and baby. This also provides an opportunity to explore management strategies to reduce risks. For this growing cohort of women, there is a great need for pre-conceptual counselling.This retrospective comparative audit assessed new referrals and pre-conceptual counselling of women attending a joint obstetric-cardiology clinic at a tertiary maternity centre in a 12-month period of 2015-2016 compared with 2018-2019. This reflected the timing of the introduction of a multidisciplinary meeting prior to clinics and assessed the impact on referrals with the introduction of the European Society of Cardiology guidelines.Data were reviewed from 56 and 67 patients in respective audit periods. Patient's risk was stratified using modified World Health Organization classification.Less than 50% of women with pre-existing cardiac conditions had received pre-conceptual counselling, although half of them had risks clearly documented. The majority of patients had a recent electrocardiograph and echocardiogram performed prior to counselling, and there was a modest improvement in the number of appropriate functional tests performed between time points. One-third of patients in both cohorts were taking cardiac medications during pregnancy.There was a significant increase in the number of pregnant women with cardiac disease and in complexity according to modified World Health Organization risk classification. While there have been improvements, it is clear that further work to improve availability and documentation of pre-pregnancy counselling is needed.
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Cardiología , Cardiopatías , Consejo , Femenino , Cardiopatías/complicaciones , Cardiopatías/epidemiología , Cardiopatías/prevención & control , Humanos , Embarazo , Resultado del Embarazo , Estudios RetrospectivosRESUMEN
BACKGROUND: Women with cardiac disease are thought to be at increased risk of post-partum haemorrhage. We sought to assess the estimated blood loss (EBL) in our cohort of women with and without cardiac disease (CD) in a quaternary hospital in the UK. Our population consisted of both congenital and acquired CD; and low risk women who delivered in our unit between 01/01/2012-30/09/2016. METHODS: Data were collected using computerised hospital records. CD was classified according to the modified WHO classification (mWHO). The primary outcome measure was estimated blood loss (mL). RESULTS: A total of 5413 women with a singleton fetus in the cephalic presentation delivered during the study period (159 women with CD and 5254 controls). In the CD group, active management of the third stage of labour was consistent with that used in low risk women in 98% (152/155) of cases. Multivariable analyses demonstrated no significant difference in EBL between women with CD vs controls. The adjusted average blood losses were 247.2 ml, 241.8 ml and 295.9 ml in the control group, mWHO 1-2 and 3-4, respectively (p = 0.165). CONCLUSIONS: Women with CD have comparable EBL to low risk women when management of the active third stage of labour is the same.
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Cardiopatías/epidemiología , Oxitócicos/uso terapéutico , Oxitocina/uso terapéutico , Hemorragia Posparto/prevención & control , Complicaciones Cardiovasculares del Embarazo/epidemiología , Adulto , Estudios de Casos y Controles , Cesárea , Estudios de Cohortes , Parto Obstétrico , Femenino , Humanos , Tercer Periodo del Trabajo de Parto , Análisis Multivariante , Embarazo , Estudios Retrospectivos , Reino Unido/epidemiología , Adulto JovenRESUMEN
[This corrects the article DOI: 10.1371/journal.pgen.1004933.].
RESUMEN
Type 1 diabetes (T1D) results from the progressive loss of pancreatic beta cells as a result of autoimmune destruction. We recently reported that during the natural history of T1D in humans and the female nonobese diabetic (NOD) mouse model, beta cells acquire a senescence-associated secretory phenotype (SASP) that is a major driver of disease onset and progression, but the mechanisms that activate SASP in beta cells were not explored. Here, we show that the SASP in islet cells is transcriptionally controlled by Bromodomain ExtraTerminal (BET) proteins, including Bromodomain containing protein 4 (BRD4). A chromatin analysis of key beta cell SASP genes in NOD islets revealed binding of BRD4 at active regulatory regions. BET protein inhibition in NOD islets diminished not only the transcriptional activation and secretion of SASP factors, but also the non-cell autonomous activity. BET protein inhibition also decreased the extent of SASP induction in human islets exposed to DNA damage. The BET protein inhibitor iBET-762 prevented diabetes in NOD mice and also attenuated SASP in islet cells in vivo. Taken together, our findings support a crucial role for BET proteins in the activation of the SASP transcriptional program in islet cells. These studies suggest avenues for preventing T1D by transcriptional inhibition of SASP.
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Proteínas de Ciclo Celular/metabolismo , Senescencia Celular/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Islotes Pancreáticos/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Animales , Diabetes Mellitus Tipo 1/inmunología , Femenino , Humanos , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/inmunología , Ratones , Ratones Endogámicos NOD , Comunicación Paracrina , Unión ProteicaRESUMEN
Retrotransposition of endogenous retroviruses (ERVs) poses a substantial threat to genome stability. Transcriptional silencing of a subset of these parasitic elements in early mouse embryonic and germ cell development is dependent upon the lysine methyltransferase SETDB1, which deposits H3K9 trimethylation (H3K9me3) and the co-repressor KAP1, which binds SETDB1 when SUMOylated. Here we identified the transcription co-factor hnRNP K as a novel binding partner of the SETDB1/KAP1 complex in mouse embryonic stem cells (mESCs) and show that hnRNP K is required for ERV silencing. RNAi-mediated knockdown of hnRNP K led to depletion of H3K9me3 at ERVs, concomitant with de-repression of proviral reporter constructs and specific ERV subfamilies, as well as a cohort of germline-specific genes directly targeted by SETDB1. While hnRNP K recruitment to ERVs is dependent upon KAP1, SETDB1 binding at these elements requires hnRNP K. Furthermore, an intact SUMO conjugation pathway is necessary for SETDB1 recruitment to proviral chromatin and depletion of hnRNP K resulted in reduced SUMOylation at ERVs. Taken together, these findings reveal a novel regulatory hierarchy governing SETDB1 recruitment and in turn, transcriptional silencing in mESCs.
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Metilación de ADN/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo K/genética , N-Metiltransferasa de Histona-Lisina/genética , Transcripción Genética , Animales , Cromatina/genética , Células Madre Embrionarias/virología , Retrovirus Endógenos/genética , Silenciador del Gen , Células Germinativas , Ratones , Ratones Noqueados , ARN Interferente Pequeño , Retroelementos , Elementos Silenciadores Transcripcionales/genética , Sumoilación/genéticaRESUMEN
Recent years have seen increased recognition for the role of ß-cell stress as a contributing factor to the autoimmune destruction process that ultimately results in symptomatic type 1 diabetes (T1D). Preclinical studies have discovered a variety of stress responses in the ß-cell that occur at presymptomatic stages and contribute to disease progression, but unifying explanations of how these mechanisms operate to promote disease progression remain incomplete. We propose that stressed ß-cells transition into ß-cells expressing inflammatory molecules that provoke an immune response to restore homeostasis by coordinating islet repair and the removal of stressed cells. However, when immune surveillance fails, stressed ß-cells accumulate and contribute to autoimmunity. Therapies directed toward stressed ß-cells to either curb their inflammatory signaling or to eliminate them (essentially doing the job of the failed immune surveillance) are moving from animal models into the clinic with promising initial results, although the understanding of how the immune response is coordinated by stressed ß-cells is not clear. In this article, we discuss ß-cell stress responses implicated in T1D pathogenesis based on evidence from humans and highlight existing knowledge gaps in their mechanisms. Future work in this field is poised to target T1D by simultaneously targeting stressed ß-cells and the failed immune response to halt the progression of autoimmunity and prevent ß-cell destruction.
RESUMEN
Type 1 diabetes (T1D) is a metabolic disease resulting from progressive autoimmune destruction of insulin-producing pancreatic beta cells. Although the majority of beta cells are lost in T1D, a small subset undergoes senescence, a stress response involving growth arrest, DNA damage response, and activation of a senescence-associated secretory phenotype (SASP). SASP in beta cells of the nonobese diabetic (NOD) mouse model of T1D and primary human islets is regulated at the level of transcription by bromodomain extra-terminal (BET) proteins, but the mechanisms remain unclear. To explore how SASP is transcriptionally regulated in beta cells, we used the NOD beta cell line NIT-1 to model beta cell SASP and identified binding partners of BET protein Brd4 and explored the role of the cyclin-dependent kinase inhibitor p21. Brd4 interacted with a variety of proteins in senescent NIT-1 cells including subunits of the Ino80 chromatin remodeling complex, which was expressed in beta cells during T1D progression in NOD mice and in human beta cells of control, autoantibody-positive, and T1D donors as determined from single-cell RNA-seq data. RNAi knockdown of p21 during senescence in NIT-1 cells did not significantly impact viability or SASP. Taken together, these results suggest that Brd4 interacts with several protein partners during senescence in NIT-1 cells, some of which may play roles in SASP gene activation and that p21 is dispensable for the SASP in this beta cell model.
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A subpopulation of pancreatic beta cells becomes senescent during type 1 diabetes (T1D) progression, and removal of these populations protects against T1D in mice. Here, we present a protocol to measure senescence in murine pancreatic islet cells through analysis of senescence-associated ß-galactosidase activity. We describe steps for staining with the fluorogenic substrate C12FDG and analysis by flow cytometry. Increased cell size is another marker of senescence and can also be concurrently measured in the same experiment. For complete details on the use and execution of this protocol, please refer to Lee et al.1 and Helman et al.2.
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Senescencia Celular , Diabetes Mellitus Tipo 1 , Ratones , Animales , beta-Galactosidasa , Células Epiteliales , Modelos Animales de EnfermedadRESUMEN
Cellular senescence is a programmed state of cell cycle arrest that involves a complex immunogenic secretome, eliciting immune surveillance and senescent cell clearance. Recent work has shown that a subpopulation of pancreatic ß-cells becomes senescent in the context of diabetes; however, it is not known whether these cells are normally subject to immune surveillance. In this opinion article, we advance the hypothesis that immune surveillance of ß-cells undergoing a senescence stress response normally limits their accumulation during aging and that the breakdown of these mechanisms is a driver of senescent ß-cell accumulation in diabetes. Elucidation and therapeutic activation of immune surveillance mechanisms in the pancreas holds promise for the improvement of approaches to target stressed senescent ß-cells in the treatment of diabetes.
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Senescencia Celular , Vigilancia Inmunológica , Células Secretoras de Insulina , Células Secretoras de Insulina/inmunología , Células Secretoras de Insulina/metabolismo , Senescencia Celular/inmunología , Senescencia Celular/fisiología , Humanos , Animales , Diabetes Mellitus/inmunologíaRESUMEN
Cellular senescence is a response to a wide variety of stressors, including DNA damage, oncogene activation and physiologic aging, and pathologically accelerated senescence contributes to human disease, including diabetes mellitus. Indeed, recent work in this field has demonstrated a role for pancreatic ß-cell senescence in the pathogenesis of Type 1 Diabetes, Type 2 Diabetes and monogenic diabetes. Small molecule or genetic targeting of senescent ß-cells has shown promise as a novel therapeutic approach for preventing and treating diabetes. Despite these advances, major questions remain around the molecular mechanisms driving senescence in the ß-cell, identification of molecular markers that distinguish senescent from non-senescent ß-cell subpopulations, and translation of proof-of-concept therapies into novel treatments for diabetes in humans. Here, we summarize the current state of the field of ß-cell senescence, highlighting insights from mouse models as well as studies on human islets and ß-cells. We identify markers that have been used to detect ß-cell senescence to unify future research efforts in this field. We discuss emerging concepts of the natural history of senescence in ß-cells, heterogeneity of senescent ß-cells subpopulations, role of sex differences in senescent responses, and the consequences of senescence on integrated islet function and microenvironment. As a young and developing field, there remain many open research questions which need to be addressed to move senescence-targeted approaches towards clinical investigation.
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Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 2 , Femenino , Masculino , Humanos , Animales , Ratones , Diabetes Mellitus Tipo 2/terapia , Envejecimiento , Senescencia Celular , Daño del ADNRESUMEN
INTRODUCTION: The Ross procedure, where a pulmonary autograft (neoaorta) replaces the aortic valve, has excellent long-term outcomes in patients with congenital aortic valve disease. However, there are reports of neoaortic dilatation and dissection. An increasing number of women are wishing to become pregnant following the Ross procedure, but little is known about the occurrence and risks of neoaortic dilatation and complications in pregnancy. We investigated neoaorta function and outcomes in pregnancy following the Ross procedure. METHODS: This retrospective study investigated women post-Ross procedure at a tertiary ACHD unit between 1997 and 2021. Imaging evaluated neoaortic root dimensions and regurgitation pre-, and post- pregnancy, compared with matched non-pregnant controls. Primary endpoints were change in neoaortic dimensions, degree of regurgitation and adverse maternal outcomes. RESULTS: Nineteen pregnancies in 12 women were included. The mean change in neoaortic root diameter post-pregnancy was 1.8 mm (SD 3.4) (p = 0.017). There was no significant change in neoaortic dimensions in matched controls during follow-up. There were no cases of dissection, arrhythmia, acute coronary syndrome, or maternal mortality. Three deliveries were pre-term, including one emergency Caesarean section due to maternal cardiac decompensation, requiring aortic root replacement post-partum but there were no neonatal deaths. CONCLUSIONS: Pregnancy following the Ross procedure is associated with neoaortic dilatation, and pregnancy is generally well tolerated. Although adverse maternal outcomes are uncommon, there are still rare cases of cardiac complications in and around the time of pregnancy. These findings emphasise the need for accessible pre-pregnancy counselling, risk stratification and careful surveillance through pregnancy by specialist cardio-obstetric multi-disciplinary teams.
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Insuficiencia de la Válvula Aórtica , Estenosis de la Válvula Aórtica , Válvula Pulmonar , Humanos , Femenino , Embarazo , Insuficiencia de la Válvula Aórtica/diagnóstico por imagen , Insuficiencia de la Válvula Aórtica/cirugía , Insuficiencia de la Válvula Aórtica/etiología , Estudios Retrospectivos , Resultado del Embarazo/epidemiología , Autoinjertos , Cesárea , Trasplante Autólogo/efectos adversos , Válvula Aórtica/diagnóstico por imagen , Válvula Aórtica/cirugía , Estenosis de la Válvula Aórtica/cirugía , Dilatación Patológica , Válvula Pulmonar/diagnóstico por imagen , Válvula Pulmonar/cirugía , Estudios de SeguimientoRESUMEN
During the progression of type 1 diabetes (T1D), ß cells are exposed to significant stress and, therefore, require adaptive responses to survive. The adaptive mechanisms that can preserve ß cell function and survival in the face of autoimmunity remain unclear. Here, we show that the deletion of the unfolded protein response (UPR) genes Atf6α or Ire1α in ß cells of non-obese diabetic (NOD) mice prior to insulitis generates a p21-driven early senescence phenotype and alters the ß cell secretome that significantly enhances the leukemia inhibitory factor-mediated recruitment of M2 macrophages to islets. Consequently, M2 macrophages promote anti-inflammatory responses and immune surveillance that cause the resolution of islet inflammation, the removal of terminally senesced ß cells, the reduction of ß cell apoptosis, and protection against T1D. We further demonstrate that the p21-mediated early senescence signature is conserved in the residual ß cells of T1D patients. Our findings reveal a previously unrecognized link between ß cell UPR and senescence that, if leveraged, may represent a novel preventive strategy for T1D.
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Diabetes Mellitus Tipo 1 , Células Secretoras de Insulina , Islotes Pancreáticos , Ratones , Animales , Humanos , Diabetes Mellitus Tipo 1/metabolismo , Endorribonucleasas/metabolismo , Ratones Endogámicos NOD , Proteínas Serina-Treonina Quinasas/metabolismo , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/metabolismoRESUMEN
Type 1 Diabetes (T1D) is caused by insulin deficiency, due to progressive autoimmune destruction of pancreatic ß cells. Glucagon-secreting α cells become dysfunctional in T1D and contribute to pathophysiology, however, the mechanisms involved are unclear. While the majority of ß cells are destroyed in T1D, some ß cells escape this fate and become senescent but whether α cell dysfunction involves a senescence program has not been explored. Here we addressed the question of whether α cells become senescent during the natural history of T1D in the non-obese diabetic (NOD) mouse model and humans. NOD mice had several distinct subpopulations of α cells, but none were defined by markers of senescence at the transcriptional or protein level. Similarly, α cells of human T1D donors did not express senescence markers. Despite the lack of senescence in α cells in vivo, using a human islet culture model, we observed that DNA damage-induced senescence led to alterations in islet glucagon secretion, which could be rescued by inhibiting the senescence-associated secretory phenotype (SASP). Together our results suggest that α cell dysfunction in T1D is not due to activation of a senescence program, however, senescent ß cell accumulation in the islet microenvironment may have a negative effect on α cell function.
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Diabetes Mellitus Tipo 1 , Células Secretoras de Glucagón , Ratones , Animales , Humanos , Diabetes Mellitus Tipo 1/metabolismo , Ratones Endogámicos NOD , Glucagón/metabolismo , Células Secretoras de Glucagón/metabolismo , Insulina/metabolismo , Biomarcadores/metabolismoRESUMEN
OBJECTIVE: Type 1 Diabetes (T1D) is characterized by progressive loss of insulin-producing pancreatic ß cells as a result of autoimmune destruction. In addition to ß cell death, recent work has shown that subpopulations of ß cells acquire dysfunction during T1D. We previously reported that ß cells undergoing a DNA damage response (DDR) and senescence accumulate during the pathogenesis of T1D. However, the question of how senescence develops in ß cells has not been investigated. METHODS: Here, we tested the hypothesis that unrepaired DNA damage in the context of genetic susceptibility triggers ß cell senescence using culture models including the mouse NIT1 ß cell line derived from the T1D-susceptible nonobese diabetic (NOD) strain, human donor islets and EndoC ß cells. DNA damage was chemically induced using etoposide or bleomycin and cells or islets were analyzed by a combination of molecular assays for senescence phenotypes including Western blotting, qRT-PCR, Luminex assays, flow cytometry and histochemical staining. RNA-seq was carried out to profile global transcriptomic changes in human islets undergoing DDR and senescence. Insulin ELISAs were used to quantify glucose-stimulated insulin secretion from chemically-induced senescent human islets, EndoC ß cells and mouse ß cell lines in culture. RESULTS: Sub-lethal DNA damage in NIT1 cells led to several classical hallmarks of senescence including sustained DDR activation, growth arrest, enlarged flattened morphology and a senescence-associated secretory phenotype (SASP) resembling what occurs in primary ß cells during T1D in NOD mice. These phenotypes differed between NIT1 cells and the MIN6 ß cell line derived from a non-T1D susceptible mouse strain. RNA-seq analysis of DNA damage-induced senescence in human islets from two different donors revealed a p53 transcriptional program and upregulation of prosurvival and SASP genes, with inter-donor variability in this response. Inter-donor variability in human islets was also apparent in the extent of persistent DDR activation and SASP at the protein level. Notably, chemically induced DNA damage also led to DDR activation and senescent phenotypes in EndoC-ßH5 human ß cells, confirming that this response can occur directly in a human ß cell line. Finally, DNA damage led to different effects on glucose-stimulated insulin secretion in mouse ß cell lines as compared with human islets and EndoC ß cells. CONCLUSIONS: Taken together, these findings suggest that some of the phenotypes of senescent ß cells that accumulate during the development of T1D in the NOD mouse and humans can be modeled by chemically induced DNA damage to mouse ß cell lines, human islets and EndoC ß cells in culture. The differences between ß cells from different mouse strains and different human islet donors and EndoC ß cells highlights species differences and the role for genetic background in modifying the ß cell response to DNA damage and its effects on insulin secretion. These culture models will be useful tools to understand some of the mechanisms of ß cell senescence in T1D.
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Diabetes Mellitus Tipo 1 , Células Secretoras de Insulina , Animales , Daño del ADN , Diabetes Mellitus Tipo 1/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Ratones , Ratones Endogámicos NODRESUMEN
BACKGROUND: The failure of immune surveillance to remove senescent cells drive age-related diseases. Here, we target an endogenous immune surveillance mechanism that can promote elimination of senescent cells and reverse disease progression. METHODS: We identify a class of lipid-activated T cells, invariant natural killer T cells (iNKTs) are involved in the removal of pathologic senescent cells. We use two disease models in which senescent cells accumulate to test whether activation of iNKT cells was sufficient to eliminate senescent cells in vivo. FINDINGS: Senescent preadipocytes accumulate in white adipose tissue of chronic high-fat diet (HFD) fed mice, and activation of iNKT cells with the prototypical glycolipid antigen alpha-galactosylceramide (αGalCer) led to a reduction of these cells with improved glucose control. Similarly, senescent cells accumulate within the lungs of mice injured by inhalational bleomycin, and αGalCer-induced activation of iNKT cells greatly limited this accumulation, decreased the lung fibrosis and improved survival. Furthermore, co-culture experiments showed that the preferential cytotoxic activity of iNKT cells to senescent cells is conserved in human cells. CONCLUSIONS: These results uncover a senolytic capacity of tissue-resident iNKT cells and pave the way for anti-senescence therapies that target these cells and their mechanism of activation.
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Células T Asesinas Naturales , Animales , Senescencia Celular , Dieta Alta en Grasa , Recuento de Linfocitos , RatonesRESUMEN
Type 1 diabetes (T1D) is a chronic metabolic disease characterized by insulin deficiency, generally resulting from progressive autoimmune-mediated destruction of pancreatic beta cells. While the phenomenon of beta cell autoimmunity continues to be an active area of investigation, recent evidence suggests that beta cell stress responses are also important contributors to disease onset. Here we review the pathways driving different kinds of beta cell dysfunction and their respective therapeutic targets in the prevention of T1D. We discuss opportunities and important open questions around the effectiveness of beta cell therapies and challenges for clinical utility. We further evaluate ways in which beta cell drug therapy could be combined with immunotherapy for preventing T1D in light of our growing appreciation of disease heterogeneity and patient endotypes. Ultimately, the emergence of pharmacologic beta cell therapies for T1D have armed us with new tools and closing the knowledge gaps in T1D etiology will be essential for maximizing the potential of these approaches.