RESUMO
Cell-identity switches, in which terminally differentiated cells are converted into different cell types when stressed, represent a widespread regenerative strategy in animals, yet they are poorly documented in mammals. In mice, some glucagon-producing pancreatic α-cells and somatostatin-producing δ-cells become insulin-expressing cells after the ablation of insulin-secreting ß-cells, thus promoting diabetes recovery. Whether human islets also display this plasticity, especially in diabetic conditions, remains unknown. Here we show that islet non-ß-cells, namely α-cells and pancreatic polypeptide (PPY)-producing γ-cells, obtained from deceased non-diabetic or diabetic human donors, can be lineage-traced and reprogrammed by the transcription factors PDX1 and MAFA to produce and secrete insulin in response to glucose. When transplanted into diabetic mice, converted human α-cells reverse diabetes and continue to produce insulin even after six months. Notably, insulin-producing α-cells maintain expression of α-cell markers, as seen by deep transcriptomic and proteomic characterization. These observations provide conceptual evidence and a molecular framework for a mechanistic understanding of in situ cell plasticity as a treatment for diabetes and other degenerative diseases.
Assuntos
Diabetes Mellitus/patologia , Diabetes Mellitus/terapia , Células Secretoras de Glucagon/citologia , Células Secretoras de Glucagon/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/patologia , Animais , Biomarcadores/análise , Linhagem da Célula/efeitos dos fármacos , Reprogramação Celular/efeitos dos fármacos , Diabetes Mellitus/imunologia , Diabetes Mellitus/metabolismo , Modelos Animais de Doenças , Feminino , Glucagon/metabolismo , Células Secretoras de Glucagon/efeitos dos fármacos , Células Secretoras de Glucagon/transplante , Glucose/farmacologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/metabolismo , Fatores de Transcrição Maf Maior/genética , Fatores de Transcrição Maf Maior/metabolismo , Masculino , Camundongos , Especificidade de Órgãos/efeitos dos fármacos , Polipeptídeo Pancreático/metabolismo , Células Secretoras de Polipeptídeo Pancreático/citologia , Células Secretoras de Polipeptídeo Pancreático/efeitos dos fármacos , Células Secretoras de Polipeptídeo Pancreático/metabolismo , Proteômica , Análise de Sequência de RNA , Transativadores/genética , Transativadores/metabolismo , Transcriptoma , Transdução GenéticaRESUMO
Total or near-total loss of insulin-producing ß-cells occurs in type 1 diabetes. Restoration of insulin production in type 1 diabetes is thus a major medical challenge. We previously observed in mice in which ß-cells are completely ablated that the pancreas reconstitutes new insulin-producing cells in the absence of autoimmunity. The process involves the contribution of islet non-ß-cells; specifically, glucagon-producing α-cells begin producing insulin by a process of reprogramming (transdifferentiation) without proliferation. Here we show the influence of age on ß-cell reconstitution from heterologous islet cells after near-total ß-cell loss in mice. We found that senescence does not alter α-cell plasticity: α-cells can reprogram to produce insulin from puberty through to adulthood, and also in aged individuals, even a long time after ß-cell loss. In contrast, before puberty there is no detectable α-cell conversion, although ß-cell reconstitution after injury is more efficient, always leading to diabetes recovery. This process occurs through a newly discovered mechanism: the spontaneous en masse reprogramming of somatostatin-producing δ-cells. The juveniles display 'somatostatin-to-insulin' δ-cell conversion, involving dedifferentiation, proliferation and re-expression of islet developmental regulators. This juvenile adaptability relies, at least in part, upon the combined action of FoxO1 and downstream effectors. Restoration of insulin producing-cells from non-ß-cell origins is thus enabled throughout life via δ- or α-cell spontaneous reprogramming. A landscape with multiple intra-islet cell interconversion events is emerging, offering new perspectives for therapy.
Assuntos
Envelhecimento/fisiologia , Transdiferenciação Celular , Diabetes Mellitus Experimental/patologia , Células Secretoras de Insulina/citologia , Insulina/biossíntese , Regeneração , Células Secretoras de Somatostatina/citologia , Animais , Desdiferenciação Celular , Proliferação de Células , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 1/terapia , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/metabolismo , Células Secretoras de Glucagon/citologia , Células Secretoras de Glucagon/metabolismo , Humanos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Camundongos , Maturidade Sexual , Somatostatina/biossíntese , Somatostatina/metabolismo , Células Secretoras de Somatostatina/metabolismoRESUMO
Using single transcription factors to reprogram cells could produce important insights into the epigenetic mechanisms that direct normal differentiation, or counter inappropriate plasticity, or even provide new ways of manipulating normal ontogeny in vitro to control lineage diversification and differentiation. We enforced Pdx1 expression from the Neurogenin-3-expressing endocrine commitment point onward and found during the embryonic period a minor increased ß-cell allocation with accompanying reduced α-cell numbers. More surprisingly, almost all remaining Pdx1-containing glucagon/Arx-producing cells underwent a fairly rapid conversion at postnatal stages, through glucagon-insulin double positivity, to a state indistinguishable from normal ß cells, resulting in complete α-cell absence. This α-to-ß conversion was not caused by activating Pdx1 in the later glucagon-expressing state. Our findings reveal that Pdx1 can work single-handedly as a potent context-dependent autonomous reprogramming agent, and suggest a postnatal differentiation evaluation stage involved in normal endocrine maturation.
Assuntos
Células Secretoras de Glucagon/metabolismo , Proteínas de Homeodomínio/metabolismo , Células Secretoras de Insulina/metabolismo , Transativadores/metabolismo , Animais , Animais Recém-Nascidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Glucagon/genética , Glucagon/metabolismo , Células Secretoras de Glucagon/citologia , Proteínas de Homeodomínio/genética , Imuno-Histoquímica , Insulina/genética , Insulina/metabolismo , Células Secretoras de Insulina/citologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Pâncreas/embriologia , Pâncreas/crescimento & desenvolvimento , Pâncreas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores/genéticaRESUMO
Pancreatic insulin-producing beta-cells have a long lifespan, such that in healthy conditions they replicate little during a lifetime. Nevertheless, they show increased self-duplication after increased metabolic demand or after injury (that is, beta-cell loss). It is not known whether adult mammals can differentiate (regenerate) new beta-cells after extreme, total beta-cell loss, as in diabetes. This would indicate differentiation from precursors or another heterologous (non-beta-cell) source. Here we show beta-cell regeneration in a transgenic model of diphtheria-toxin-induced acute selective near-total beta-cell ablation. If given insulin, the mice survived and showed beta-cell mass augmentation with time. Lineage-tracing to label the glucagon-producing alpha-cells before beta-cell ablation tracked large fractions of regenerated beta-cells as deriving from alpha-cells, revealing a previously disregarded degree of pancreatic cell plasticity. Such inter-endocrine spontaneous adult cell conversion could be harnessed towards methods of producing beta-cells for diabetes therapies, either in differentiation settings in vitro or in induced regeneration.
Assuntos
Diferenciação Celular/fisiologia , Transdiferenciação Celular/fisiologia , Células Secretoras de Glucagon/citologia , Células Secretoras de Insulina/citologia , Animais , Biomarcadores/metabolismo , Contagem de Células , Morte Celular/efeitos dos fármacos , Linhagem da Célula , Proliferação de Células , Reprogramação Celular , Toxina Diftérica/farmacologia , Toxina Diftérica/toxicidade , Feminino , Glucagon/biossíntese , Glucagon/genética , Glucagon/metabolismo , Células Secretoras de Glucagon/metabolismo , Humanos , Insulina/biossíntese , Insulina/metabolismo , Insulina/farmacologia , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Ratos , Regeneração/fisiologiaRESUMO
All pancreatic endocrine cell types arise from a common endocrine precursor cell population, yet the molecular mechanisms that establish and maintain the unique gene expression programs of each endocrine cell lineage have remained largely elusive. Such knowledge would improve our ability to correctly program or reprogram cells to adopt specific endocrine fates. Here, we show that the transcription factor Nkx6.1 is both necessary and sufficient to specify insulin-producing beta cells. Heritable expression of Nkx6.1 in endocrine precursors of mice is sufficient to respecify non-beta endocrine precursors towards the beta cell lineage, while endocrine precursor- or beta cell-specific inactivation of Nkx6.1 converts beta cells to alternative endocrine lineages. Remaining insulin(+) cells in conditional Nkx6.1 mutants fail to express the beta cell transcription factors Pdx1 and MafA and ectopically express genes found in non-beta endocrine cells. By showing that Nkx6.1 binds to and represses the alpha cell determinant Arx, we identify Arx as a direct target of Nkx6.1. Moreover, we demonstrate that Nkx6.1 and the Arx activator Isl1 regulate Arx transcription antagonistically, thus establishing competition between Isl1 and Nkx6.1 as a critical mechanism for determining alpha versus beta cell identity. Our findings establish Nkx6.1 as a beta cell programming factor and demonstrate that repression of alternative lineage programs is a fundamental principle by which beta cells are specified and maintained. Given the lack of Nkx6.1 expression and aberrant activation of non-beta endocrine hormones in human embryonic stem cell (hESC)-derived insulin(+) cells, our study has significant implications for developing cell replacement therapies.
Assuntos
Células Endócrinas , Proteínas de Homeodomínio , Células Secretoras de Insulina , Insulina , Animais , Diferenciação Celular/genética , Linhagem da Célula , Terapia Baseada em Transplante de Células e Tecidos , Células Endócrinas/citologia , Células Endócrinas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Insulina/genética , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Fatores de Transcrição Maf Maior/genética , Fatores de Transcrição Maf Maior/metabolismo , Camundongos , Pâncreas/citologia , Células-Tronco , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
AIMS/HYPOTHESIS: Non-invasive imaging of beta cells is a much-needed development but is one that faces significant biological and technological hurdles. A relevant imaging method should at least allow for an evaluation over time of the mass of beta cells under physiological and pathological conditions, and for an assessment of novel therapies. We, therefore, investigated the ability of a new MRI probe to repeatedly measure the loss of beta cells in a rodent model. METHODS: We developed an innovative nanoparticle probe that targets the glucagon-like peptide 1 receptor, and can be used for both fluorescence imaging and MRI. Using fluorescence, we characterised the specificity and biodistribution of the probe. Using 1.5 T MRI, we longitudinally imaged the changes in insulin content in male and female mice of the RIP-DTr strain, which mimic the changes expected in type 1 and type 2 diabetes, respectively. RESULTS: We showed that this probe selectively labelled beta cells in situ, imaged in vivo native pancreatic islets and evaluated their loss after diphtheria toxin administration, in a model of graded beta cell deletion. Thus, using clinical MRI, the probe quantitatively differentiates, in the same mouse strain, between female animals featuring a 50% loss of beta cells and the males featuring an almost complete loss of beta cells. CONCLUSIONS/INTERPRETATION: The approach addresses several of the hurdles that have so far limited the non-invasive imaging of beta cells, including the potential to repeatedly monitor the very same animals using clinically available equipment, and to differentiate graded losses of beta cells.
Assuntos
Glucagon/metabolismo , Células Secretoras de Insulina/metabolismo , Imageamento por Ressonância Magnética , Fragmentos de Peptídeos/metabolismo , Receptores de Glucagon/metabolismo , Animais , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Sondas Moleculares , Distribuição TecidualRESUMO
Insulin-producing ß-cells in pancreatic islets are regulated by systemic cues and, locally, by adjacent islet hormone-producing 'non-ß-cells' (namely α-cells, δ-cells and γ-cells). Yet whether the non-ß-cells are required for accurate insulin secretion is unclear. Here, we studied mice in which adult islets are exclusively composed of ß-cells and human pseudoislets containing only primary ß-cells. Mice lacking non-ß-cells had optimal blood glucose regulation, enhanced glucose tolerance, insulin sensitivity and restricted body weight gain under a high-fat diet. The insulin secretion dynamics in islets composed of only ß-cells was comparable to that in intact islets. Similarly, human ß-cell pseudoislets retained the glucose-regulated mitochondrial respiration, insulin secretion and exendin-4 responses of entire islets. The findings indicate that non-ß-cells are dispensable for blood glucose homeostasis and ß-cell function. These results support efforts aimed at developing diabetes treatments by generating ß-like clusters devoid of non-ß-cells, such as from pluripotent stem cells differentiated in vitro or by reprograming non-ß-cells into insulin producers in situ.
RESUMO
GNAO1 encephalopathy characterized by a wide spectrum of neurological deficiencies in pediatric patients originates from de novo heterozygous mutations in the gene encoding Gαo, the major neuronal G protein. Efficient treatments and even the proper understanding of the underlying etiology are currently lacking for this dominant disease. Adequate animal models of GNAO1 encephalopathy are urgently needed. Here we describe establishment and characterization of mouse models of the disease based on two point mutations in GNAO1 with different clinical manifestations. One of them is G203R leading to the early-onset epileptic seizures, motor dysfunction, developmental delay and intellectual disability. The other is C215Y producing much milder clinical outcomes, mostly-late-onset hyperkinetic movement disorder. The resultant mouse models show distinct phenotypes: severe neonatal lethality in GNAO1[G203R]/ + mice vs. normal vitality in GNAO1[C215Y]/ + . The latter model further revealed strong hyperactivity and hyperlocomotion in a panel of behavioral assays, without signs of epilepsy, recapitulating the patients' manifestations. Importantly, despite these differences the two models similarly revealed prenatal brain developmental anomalies, such as enlarged lateral ventricles and decreased numbers of neuronal precursor cells in the cortex. Thus, our work unveils GNAO1 encephalopathy as to a large extent neurodevelopmental malady. We expect that this understanding and the tools we established will be instrumental for future therapeutic developments.
Assuntos
Encefalopatias/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Transtornos do Neurodesenvolvimento/genética , Adolescente , Adulto , Idoso , Animais , Criança , Pré-Escolar , Modelos Animais de Doenças , Epilepsia/genética , Feminino , Humanos , Masculino , Camundongos , Mutação , FenótipoRESUMO
The precise developmental dynamics of the pancreatic islet endocrine cell types, and their interrelation, are unknown. Some authors claim the persistence of islet cell differentiation from precursor cells after birth ("neogenesis"). Here, using four conditional cell lineage tracing ("pulse-and-chase") murine models, we describe the natural history of pancreatic islet cells, once they express a hormone gene, until late in life. Concerning the contribution of early-appearing embryonic hormone-expressing cells to the formation of islets, we report that adult islet cells emerge from embryonic hormone-expressing cells arising at different time points during development, without any evidence of postnatal neogenesis. We observe specific patterns of hormone gene activation and switching during islet morphogenesis, revealing that, within each cell type, cells have heterogeneous developmental trajectories. This likely applies to most maturating cells in the body, and explains the observed phenotypic variability within differentiated cell types. Such knowledge should help devising novel regenerative therapies.
Assuntos
Envelhecimento/fisiologia , Feto/citologia , Hormônios/metabolismo , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/embriologia , Animais , Doxiciclina/farmacologia , Desenvolvimento Embrionário/efeitos dos fármacos , Feto/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Glucagon/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Camundongos Transgênicos , Somatostatina/metabolismo , Coloração e RotulagemRESUMO
Generation of surrogate cells with stable functional identities is crucial for developing cell-based therapies. Efforts to produce insulin-secreting replacement cells to treat diabetes require reliable tools to assess islet cellular identity. Here, we conduct a thorough single-cell transcriptomics meta-analysis to identify robustly expressed markers used to build genesets describing the identity of human α-, ß-, γ- and δ-cells. These genesets define islet cellular identities better than previously published genesets. We show their efficacy to outline cell identity changes and unravel some of their underlying genetic mechanisms, whether during embryonic pancreas development or in experimental setups aiming at developing glucose-responsive insulin-secreting cells, such as pluripotent stem-cell differentiation or in adult islet cell reprogramming protocols. These islet cell type-specific genesets represent valuable tools that accurately benchmark gain and loss in islet cell identity traits.
Assuntos
Células Secretoras de Insulina , Ilhotas Pancreáticas , Células-Tronco Pluripotentes , Diferenciação Celular/genética , Humanos , Insulina/genéticaRESUMO
Developmental genes are frequently controlled by multiple enhancers sharing similar specificities. As a result, deletions of such regulatory elements have often failed to reveal their full function. Here, we use the Pitx1 testbed locus to characterize in detail the regulatory and cellular identity alterations following the deletion of one of its enhancers (Pen). By combining single cell transcriptomics and an in-embryo cell tracing approach, we observe an increased fraction of Pitx1 non/low-expressing cells and a decreased fraction of Pitx1 high-expressing cells. We find that the over-representation of Pitx1 non/low-expressing cells originates from a failure of the Pitx1 locus to coordinate enhancer activities and 3D chromatin changes. This locus mis-activation induces a localized heterochrony and a concurrent loss of irregular connective tissue, eventually leading to a clubfoot phenotype. This data suggests that, in some cases, redundant enhancers may be used to locally enforce a robust activation of their host regulatory landscapes.
Assuntos
Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição Box Pareados/genética , Acetilação , Animais , Cromatina/química , Cromatina/metabolismo , Tecido Conjuntivo/crescimento & desenvolvimento , Tecido Conjuntivo/metabolismo , Embrião de Mamíferos , Epigênese Genética , Membro Posterior/citologia , Membro Posterior/embriologia , Membro Posterior/metabolismo , Botões de Extremidades/citologia , Botões de Extremidades/embriologia , Botões de Extremidades/metabolismo , Camundongos , Modelos Genéticos , Fatores de Transcrição Box Pareados/metabolismo , Deleção de SequênciaRESUMO
The cellular identity of pancreatic polypeptide (Ppy)-expressing γ-cells, one of the rarest pancreatic islet cell-type, remains elusive. Within islets, glucagon and somatostatin, released respectively from α- and δ-cells, modulate the secretion of insulin by ß-cells. Dysregulation of insulin production raises blood glucose levels, leading to diabetes onset. Here, we present the genetic signature of human and mouse γ-cells. Using different approaches, we identified a set of genes and pathways defining their functional identity. We found that the γ-cell population is heterogeneous, with subsets of cells producing another hormone in addition to Ppy. These bihormonal cells share identity markers typical of the other islet cell-types. In mice, Ppy gene inactivation or conditional γ-cell ablation did not alter glycemia nor body weight. Interestingly, upon ß-cell injury induction, γ-cells exhibited gene expression changes and some of them engaged insulin production, like α- and δ-cells. In conclusion, we provide a comprehensive characterization of γ-cells and highlight their plasticity and therapeutic potential.
Assuntos
Insulina/biossíntese , Células Secretoras de Polipeptídeo Pancreático/metabolismo , Polipeptídeo Pancreático/metabolismo , Precursores de Proteínas/metabolismo , Animais , Glicemia/metabolismo , Peso Corporal , Linhagem da Célula/genética , Feminino , Técnicas de Introdução de Genes , Humanos , Células Secretoras de Insulina/classificação , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Pâncreas/citologia , Pâncreas/embriologia , Pâncreas/crescimento & desenvolvimento , Polipeptídeo Pancreático/deficiência , Polipeptídeo Pancreático/genética , Células Secretoras de Polipeptídeo Pancreático/classificação , Células Secretoras de Polipeptídeo Pancreático/citologia , Gravidez , RNA-SeqRESUMO
BACKGROUND & AIMS: The pancreatic mass is determined by the coordinated expansion and differentiation of progenitor cells and is maintained via tight control of cell replacement rates. The basic helix-loop-helix transcription factor c-Myc is one of the main regulators of these processes in many organs. We studied the requirement of c-Myc in controlling the generation and maintenance of pancreatic mass. METHODS: We conditionally inactivated c-Myc in Pdx1+ pancreatic progenitor cells. Pancreata of mice lacking c-Myc (c-Myc(P-/-) mice) were analyzed during development and ageing. RESULTS: Pancreatic growth in c-Myc(P-/-) mice was impaired starting on E12.5, in early primordia, because of decreased proliferation and altered differentiation of exocrine progenitors; islet progenitors were spared. Acinar cell maturation was defective in the adult hypotrophic pancreas, which hampered exocrine mass maintenance in aged animals. From 2 to 10 months of age, the c-Myc(P-/-) pancreas was progressively remodeled without inflammatory injury. Loss of acinar cells increased with time, concomitantly with adipose tissue accumulation. Using a genetic cell lineage tracing analysis, we demonstrated that pancreatic adipose cells were derived directly from transdifferentiating acinar cells. This epithelial-to-mesenchyme transition was also observed in normal aged specimens and in pancreatitis. CONCLUSIONS: These results provide evidence indicating that c-Myc activity is required for growth and maturation of the exocrine pancreas, and sheds new light on the ontogeny of pancreatic adipose cells in processes of organ degenerescence and tissue involution.
Assuntos
Adipócitos/citologia , Pâncreas Exócrino/patologia , Proteínas Proto-Oncogênicas c-myc/fisiologia , Animais , Diferenciação Celular , Proliferação de Células , Epitélio/patologia , Proteínas de Homeodomínio/análise , Humanos , Mesoderma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-myc/análise , Transativadores/análiseRESUMO
FEN-1 and XPG are members of the FEN-1 family of structure-specific nucleases, which share a conserved active site. FEN-1 plays a central role in DNA replication, whereas XPG is involved in nucleotide excision repair (NER). Both FEN-1 and XPG are active on flap structures, but only XPG cleaves bubble substrates. The spacer region of XPG is dispensable for nuclease activity on flap substrates but is required for NER activity and for efficient processing of bubble substrates. Here, we inserted the spacer region of XPG between the nuclease domains of FEN-1 to test whether this domain would be sufficient to confer XPG-like substrate specificity and NER activity on a related nuclease. The resulting FEN-1-XPG hybrid protein is active on flap and, albeit at low levels, on bubble substrates. Like FEN-1, the activity of FEN-1-XPG was stimulated by a double-flap substrate containing a 1-nt 3' flap, whereas XPG does not show this substrate preference. Although no NER activity was detected in vitro, the FEN-1-XPG hybrid displays substantial NER activity in vivo. Hence, insertion of the XPG spacer region into FEN-1 results in a hybrid protein with biochemical properties reminiscent of both nucleases, including partial NER activity.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Endonucleases/química , Endonucleases/metabolismo , Endonucleases Flap/química , Endonucleases Flap/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Células Cultivadas , Dano ao DNA , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Endonucleases Flap/genética , Humanos , Proteínas Nucleares/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Fatores de Transcrição/genética , Raios UltravioletaRESUMO
Type 1 diabetes (T1D) results from the autoimmune destruction of insulin-producing ß cells. A comprehensive picture of the changes during T1D development is lacking due to limited sample availability, inability to sample longitudinally, and the paucity of technologies enabling comprehensive tissue profiling. Here, we analyzed 1,581 islets from 12 human donors, including eight with T1D, using imaging mass cytometry (IMC). IMC enabled simultaneous measurement of 35 biomarkers with single-cell and spatial resolution. We performed pseudotime analysis of islets through T1D progression from snapshot data to reconstruct the evolution of ß cell loss and insulitis. Our analyses revealed that ß cell destruction is preceded by a ß cell marker loss and by recruitment of cytotoxic and helper T cells. The approaches described herein demonstrate the value of IMC for improving our understanding of T1D pathogenesis, and our data lay the foundation for hypothesis generation and follow-on experiments.
Assuntos
Biomarcadores/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Citometria por Imagem/métodos , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Pâncreas/metabolismo , Progressão da Doença , Humanos , Células Secretoras de Insulina/patologia , Ilhotas Pancreáticas/patologia , Pâncreas/patologiaRESUMO
The mechanisms that restrict regeneration and maintain cell identity following injury are poorly characterized in higher vertebrates. Following ß-cell loss, 1-2% of the glucagon-producing α-cells spontaneously engage in insulin production in mice. Here we explore the mechanisms inhibiting α-cell plasticity. We show that adaptive α-cell identity changes are constrained by intra-islet insulin- and Smoothened-mediated signalling, among others. The combination of ß-cell loss or insulin-signalling inhibition, with Smoothened inactivation in α- or δ-cells, stimulates insulin production in more α-cells. These findings suggest that the removal of constitutive 'brake signals' is crucial to neutralize the refractoriness to adaptive cell-fate changes. It appears that the maintenance of cell identity is an active process mediated by repressive signals, which are released by neighbouring cells and curb an intrinsic trend of differentiated cells to change.
Assuntos
Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Transdução de Sinais , Receptor Smoothened/metabolismo , Animais , Diferenciação Celular , Plasticidade Celular , Proliferação de Células , Feminino , Células Secretoras de Glucagon/citologia , Células Secretoras de Glucagon/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Camundongos Transgênicos , Receptor Smoothened/genéticaRESUMO
XPG is the human endonuclease that cuts 3' to DNA lesions during nucleotide excision repair. Missense mutations in XPG can lead to xeroderma pigmentosum (XP), whereas truncated or unstable XPG proteins cause Cockayne syndrome (CS), normally yielding life spans of <7 years. One XP-G individual who had advanced XP/CS symptoms at 28 years has been identified. The genetic, biochemical, and cellular defects in this remarkable case provide insight into the onset of XP and CS, and they reveal a previously unrecognized property of XPG. Both of this individual's XPG alleles produce a severely truncated protein, but an infrequent alternative splice generates an XPG protein lacking seven internal amino acids, which can account for his very slight cellular UV resistance. Deletion of XPG amino acids 225 to 231 does not abolish structure-specific endonuclease activity. Instead, this region is essential for interaction with TFIIH and for the stable recruitment of XPG to sites of local UV damage after the prior recruitment of TFIIH. These results define a new functional domain of XPG, and they demonstrate that recruitment of DNA repair proteins to sites of damage does not necessarily lead to productive repair reactions. This observation has potential implications that extend beyond nucleotide excision repair.
Assuntos
Dano ao DNA/efeitos da radiação , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição TFII/metabolismo , Raios Ultravioleta , Processamento Alternativo , Sequência de Aminoácidos , Linhagem Celular , Linhagem Celular Transformada , Transformação Celular Viral , Análise Mutacional de DNA , Reparo do DNA , Proteínas de Ligação a DNA/genética , Endonucleases/análise , Endonucleases/metabolismo , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Técnica Indireta de Fluorescência para Anticorpo , Mutação da Fase de Leitura , Humanos , Immunoblotting , Lentivirus/genética , Longevidade , Masculino , Microscopia de Fluorescência , Proteínas Nucleares , Testes de Precipitina , Estrutura Terciária de Proteína , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Fator de Transcrição TFIIH , Fatores de Transcrição , Xeroderma Pigmentoso/diagnóstico , Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/metabolismo , Xeroderma Pigmentoso/patologiaRESUMO
In addition to ß-cells, pancreatic islets contain α- and δ-cells, which respectively produce glucagon and somatostatin. The reprogramming of these two endocrine cell types into insulin producers, as observed after a massive ß-cell ablation in mice, may help restoring a functional ß-cell mass in type 1 diabetes. Yet, the spontaneous α-to-ß and δ-to-ß conversion processes are relatively inefficient in adult animals and the underlying epigenetic mechanisms remain unclear. Several studies indicate that the conserved chromatin modifiers DNA methyltransferase 1 (Dnmt1) and Enhancer of zeste homolog 2 (Ezh2) are important for pancreas development and restrict islet cell plasticity. Here, to investigate the role of these two enzymes in α- and δ-cell development and fate maintenance, we genetically inactivated them in each of these two cell types. We found that loss of Dnmt1 does not enhance the conversion of α- or δ-cells toward a ß-like fate. In addition, while Dnmt1 was dispensable for the development of these two cell types, we noticed a gradual loss of α-, but not δ-cells in adult mice. Finally, we found that Ezh2 inactivation does not enhance α-cell plasticity, and, contrary to what is observed in ß-cells, does not impair α-cell proliferation. Our results indicate that both Dnmt1 and Ezh2 play distinct roles in the different islet cell types.
Assuntos
DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Células Secretoras de Glucagon/metabolismo , Homeostase , Células Secretoras de Somatostatina/metabolismo , Animais , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/deficiência , Proteína Potenciadora do Homólogo 2 de Zeste/deficiência , Ativação Enzimática , Células Secretoras de Glucagon/citologia , Camundongos , Células Secretoras de Somatostatina/citologiaRESUMO
Insulin-producing pancreatic ß cells in mice can slowly regenerate from glucagon-producing α cells in settings like ß cell loss, but the basis of this conversion is unknown. Moreover, it remains unclear if this intra-islet cell conversion is relevant to diseases like type 1 diabetes (T1D). We show that the α cell regulators Aristaless-related homeobox (Arx) and DNA methyltransferase 1 (Dnmt1) maintain α cell identity in mice. Within 3 months of Dnmt1 and Arx loss, lineage tracing and single-cell RNA sequencing revealed extensive α cell conversion into progeny resembling native ß cells. Physiological studies demonstrated that converted α cells acquire hallmark ß cell electrophysiology and show glucose-stimulated insulin secretion. In T1D patients, subsets of glucagon-expressing cells show loss of DNMT1 and ARX and produce insulin and other ß cell factors, suggesting that DNMT1 and ARX maintain α cell identity in humans. Our work reveals pathways regulated by Arx and Dnmt1 that are sufficient for achieving targeted generation of ß cells from adult pancreatic α cells.