RESUMO
AIMS/HYPOTHESIS: Recovering functional beta cell mass is a promising approach for future diabetes therapies. The aim of the present study is to investigate the effects of adjudin, a small molecule identified in a beta cell screen using zebrafish, on pancreatic beta cells and diabetes conditions in mice and human spheroids. METHODS: In zebrafish, insulin expression was examined by bioluminescence and quantitative real-time PCR (qPCR), glucose levels were examined by direct measurements and distribution using a fluorescent glucose analogue, and calcium activity in beta cells was analysed by in vivo live imaging. Pancreatic islets of wild-type postnatal day 0 (P0) and 3-month-old (adult) mice, as well as adult db/db mice (i.e. BKS(D)-Leprdb/JOrlRj), were cultured in vitro and analysed by qPCR, glucose stimulated insulin secretion and whole mount staining. RNA-seq was performed for islets of P0 and db/db mice. For in vivo assessment, db/db mice were treated with adjudin and subjected to analysis of metabolic variables and islet cells. Glucose consumption was examined in primary human hepatocyte spheroids. RESULTS: Adjudin treatment increased insulin expression and calcium response to glucose in beta cells and decreased glucose levels after beta cell ablation in zebrafish. Adjudin led to improved beta cell function, decreased beta cell proliferation and glucose responsive insulin secretion by decreasing basal insulin secretion in in vitro cultured newborn mouse islets. RNA-seq of P0 islets indicated that adjudin treatment resulted in increased glucose metabolism and mitochondrial function, as well as downstream signalling pathways involved in insulin secretion. In islets from db/db mice cultured in vitro, adjudin treatment strengthened beta cell identity and insulin secretion. RNA-seq of db/db islets indicated adjudin-upregulated genes associated with insulin secretion, membrane ion channel activity and exocytosis. Moreover, adjudin promoted glucose uptake in the liver of zebrafish in an insulin-independent manner, and similarly promoted glucose consumption in primary human hepatocyte spheroids with insulin resistance. In vivo studies using db/db mice revealed reduced nonfasting blood glucose, improved glucose tolerance and strengthened beta cell identity after adjudin treatment. CONCLUSIONS/INTERPRETATION: Adjudin promoted functional maturation of immature islets, improved function of dysfunctional islets, stimulated glucose uptake in liver and improved glucose homeostasis in db/db mice. Thus, the multifunctional drug adjudin, previously studied in various contexts and conditions, also shows promise in the management of diabetic states. DATA AVAILABILITY: Raw and processed RNA-seq data for this study have been deposited in the Gene Expression Omnibus under accession number GSE235398 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE235398 ).
Assuntos
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Ilhotas Pancreáticas , Camundongos , Humanos , Animais , Recém-Nascido , Peixe-Zebra , Diabetes Mellitus Tipo 2/metabolismo , Cálcio/metabolismo , Ilhotas Pancreáticas/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Homeostase , Fígado/metabolismoRESUMO
Regenerating pancreatic ß-cells is a potential curative approach for diabetes. We previously identified the small molecule CID661578 as a potent inducer of ß-cell regeneration, but its target and mechanism of action have remained unknown. We now screened 257 million yeast clones and determined that CID661578 targets MAP kinase-interacting serine/threonine kinase 2 (MNK2), an interaction we genetically validated in vivo. CID661578 increased ß-cell neogenesis from ductal cells in zebrafish, neonatal pig islet aggregates and human pancreatic ductal organoids. Mechanistically, we found that CID661578 boosts protein synthesis and regeneration by blocking MNK2 from binding eIF4G in the translation initiation complex at the mRNA cap. Unexpectedly, this blocking activity augmented eIF4E phosphorylation depending on MNK1 and bolstered the interaction between eIF4E and eIF4G, which is necessary for both hypertranslation and ß-cell regeneration. Taken together, our findings demonstrate a targetable role of MNK2-controlled translation in ß-cell regeneration, a role that warrants further investigation in diabetes.
Assuntos
Fator de Iniciação 4E em Eucariotos , Fator de Iniciação Eucariótico 4G , Animais , Linhagem Celular , Fator de Iniciação 4E em Eucariotos/química , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Humanos , Recém-Nascido , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Peixe-Zebra/metabolismoRESUMO
This corrects the article DOI: 10.1038/nature18614.
RESUMO
Impaired formation of the intrahepatic biliary network leads to cholestatic liver diseases, which are frequently associated with autoimmune disorders. Using a chemical mutagenesis strategy in zebrafish combined with computational network analysis, we screened for novel genes involved in intrahepatic biliary network formation. We positionally cloned a mutation in the nckap1l gene, which encodes a cytoplasmic adaptor protein for the WAVE regulatory complex. The mutation is located in the last exon after the stop codon of the primary splice isoform, only disrupting a previously unannotated minor splice isoform, which indicates that the minor splice isoform is responsible for the intrahepatic biliary network phenotype. CRISPR/Cas9-mediated nckap1l deletion, which disrupts both the primary and minor isoforms, showed the same defects. In the liver of nckap1l mutant larvae, WAVE regulatory complex component proteins are degraded specifically in biliary epithelial cells, which line the intrahepatic biliary network, thus disrupting the actin organization of these cells. We further show that nckap1l genetically interacts with the Cdk5 pathway in biliary epithelial cells. These data together indicate that although nckap1l was previously considered to be a hematopoietic cell lineage-specific protein, its minor splice isoform acts in biliary epithelial cells to regulate intrahepatic biliary network formation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Processamento Alternativo , Ductos Biliares Intra-Hepáticos/embriologia , Ductos Biliares Intra-Hepáticos/metabolismo , Morfogênese/genética , Alelos , Animais , Animais Geneticamente Modificados , Quinase 5 Dependente de Ciclina/genética , Quinase 5 Dependente de Ciclina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Ordem dos Genes , Testes Genéticos , Variação Genética , Fígado/metabolismo , Modelos Biológicos , Mutação , Fenótipo , Isoformas de RNA , Peixe-Zebra , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Vascular and haematopoietic cells organize into specialized tissues during early embryogenesis to supply essential nutrients to all organs and thus play critical roles in development and disease. At the top of the haemato-vascular specification cascade lies cloche, a gene that when mutated in zebrafish leads to the striking phenotype of loss of most endothelial and haematopoietic cells and a significant increase in cardiomyocyte numbers. Although this mutant has been analysed extensively to investigate mesoderm diversification and differentiation and continues to be broadly used as a unique avascular model, the isolation of the cloche gene has been challenging due to its telomeric location. Here we used a deletion allele of cloche to identify several new cloche candidate genes within this genomic region, and systematically genome-edited each candidate. Through this comprehensive interrogation, we succeeded in isolating the cloche gene and discovered that it encodes a PAS-domain-containing bHLH transcription factor, and that it is expressed in a highly specific spatiotemporal pattern starting during late gastrulation. Gain-of-function experiments show that it can potently induce endothelial gene expression. Epistasis experiments reveal that it functions upstream of etv2 and tal1, the earliest expressed endothelial and haematopoietic transcription factor genes identified to date. A mammalian cloche orthologue can also rescue blood vessel formation in zebrafish cloche mutants, indicating a highly conserved role in vertebrate vasculogenesis and haematopoiesis. The identification of this master regulator of endothelial and haematopoietic fate enhances our understanding of early mesoderm diversification and may lead to improved protocols for the generation of endothelial and haematopoietic cells in vivo and in vitro.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células Sanguíneas/citologia , Células Sanguíneas/metabolismo , Diferenciação Celular/genética , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Vasos Sanguíneos/citologia , Vasos Sanguíneos/embriologia , Vasos Sanguíneos/metabolismo , Sequência Conservada , Epistasia Genética , Deleção de Genes , Sequências Hélice-Alça-Hélice , Hematopoese , Mesoderma/citologia , Mesoderma/embriologia , Mesoderma/metabolismo , Mutação , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/genética , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: Experimental animal studies suggest a novel role for the folate receptor 1 in ß-cell differentiation in the pancreas, with potential implications for glycemic control. We tested the hypothesis of a protective association between prenatal folic acid use and neonatal diabetes or hyperglycemia and type 1 diabetes in an observational cohort study using data from the national population health registers in Norway. METHODS: All singleton pregnancies resulting in live births from 2005 to 2018 were identified. Prenatal exposure to folic acid was determined based on maternal report at antenatal care in early pregnancy. Diagnoses of neonatal diabetes, hyperglycemia, and type 1 diabetes for the children were identified. Associations were estimated with logistic regression or Cox proportional hazard model and included crude and adjusted estimates. RESULTS: Among 781,567 children, 69% had prenatal exposure to folic acid, 264 were diagnosed with neonatal diabetes or hyperglycemia, and 1390 with type 1 diabetes. Compared to children with no prenatal exposure to folic acid, children with prenatal exposure to folic acid had similar odds of having a neonatal diabetes or hyperglycemia diagnosis (adjusted odds ratio 0.95, 95% confidence interval [CI] 0.72, 1.25) and similar risk of being diagnosed with type 1 diabetes (adjusted hazard ratio 1.05, 95% CI 0.93, 1.18). CONCLUSIONS: No association between prenatal folic acid exposure and neonatal diabetes/hyperglycemia or type 1 diabetes was found. These findings do not rule out a translational effect of the experimental results and future studies with longer follow-up and more precise information on the window of prenatal exposure are needed.
Assuntos
Diabetes Mellitus Tipo 1/epidemiologia , Ácido Fólico/administração & dosagem , Hiperglicemia/epidemiologia , Doenças do Recém-Nascido/epidemiologia , Adulto , Índice de Massa Corporal , Estudos de Coortes , Escolaridade , Feminino , Seguimentos , Humanos , Recém-Nascido , Troca Materno-Fetal , Pessoa de Meia-Idade , Noruega/epidemiologia , Gravidez , Sistema de Registros , Fatores de Risco , Fumar/epidemiologiaRESUMO
Stem cells are defined by their capacities to self-renew and generate progeny of multiple lineages. The transcription factor SOX2 has key roles in the regulation of stem cell characteristics, but whether SOX2 achieves these functions through similar mechanisms in distinct stem cell populations is not known. To address this question, we performed RNA-seq and SOX2 ChIP-seq on embryonic mouse cortex, spinal cord, stomach and lung/esophagus. We demonstrate that, although SOX2 binds a similar motif in the different cell types, its target regions are primarily cell-type-specific and enriched for the distinct binding motifs of appropriately expressed interacting co-factors. Furthermore, cell-type-specific SOX2 binding in endodermal and neural cells is most often found around genes specifically expressed in the corresponding tissue. Consistent with this, we demonstrate that SOX2 target regions can act as cis-regulatory modules capable of directing reporter expression to appropriate tissues in a zebrafish reporter assay. In contrast, SOX2 binding sites found in both endodermal and neural tissues are associated with genes regulating general stem cell features, such as proliferation. Notably, we provide evidence that SOX2 regulates proliferation through conserved mechanisms and target genes in both germ layers examined. Together, these findings demonstrate how SOX2 simultaneously regulates cell-type-specific, as well as core transcriptional programs in neural and endodermal stem cells.
Assuntos
Sistema Nervoso Central/embriologia , Endoderma/citologia , Endoderma/embriologia , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/fisiologia , Células-Tronco Neurais/fisiologia , Organogênese/genética , Fatores de Transcrição SOXB1/fisiologia , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Sistema Nervoso Central/citologia , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Transgênicos , Células-Tronco Neurais/citologia , Regiões Promotoras Genéticas , Ligação Proteica , Fatores de Transcrição SOXB1/genéticaRESUMO
There is great interest in therapeutically harnessing endogenous regenerative mechanisms to increase the number of ß cells in people with diabetes. By performing whole-genome expression profiling of zebrafish islets, we identified 11 secreted proteins that are upregulated during ß-cell regeneration. We then tested the proteins' ability to potentiate ß-cell regeneration in zebrafish at supraphysiological levels. One protein, insulin-like growth factor (Igf) binding-protein 1 (Igfbp1), potently promoted ß-cell regeneration by potentiating α- to ß-cell transdifferentiation. Using various inhibitors and activators of the Igf pathway, we show that Igfbp1 exerts its regenerative effect, at least partly, by inhibiting Igf signaling. Igfbp1's effect on transdifferentiation appears conserved across species: Treating mouse and human islets with recombinant IGFBP1 in vitro increased the number of cells co-expressing insulin and glucagon threefold. Moreover, a prospective human study showed that having high IGFBP1 levels reduces the risk of developing type-2 diabetes by more than 85%. Thus, we identify IGFBP1 as an endogenous promoter of ß-cell regeneration and highlight its clinical importance in diabetes.
Assuntos
Transdiferenciação Celular , Células Secretoras de Glucagon/fisiologia , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Células Secretoras de Insulina/fisiologia , Regeneração , Animais , Humanos , Camundongos , Peixe-ZebraRESUMO
Spatially distinct gene expression profiles in neural stem cells (NSCs) are a prerequisite to the formation of neuronal diversity, but how these arise from the regulatory interactions between chromatin accessibility and transcription factor activity has remained unclear. Here, we demonstrate that, despite their distinct gene expression profiles, NSCs of the mouse cortex and spinal cord share the majority of their DNase I hypersensitive sites (DHSs). Regardless of this similarity, domain-specific gene expression is highly correlated with the relative accessibility of associated DHSs, as determined by sequence read density. Notably, the binding pattern of the general NSC transcription factor SOX2 is also largely cell type specific and coincides with an enrichment of LHX2 motifs in the cortex and HOXA9 motifs in the spinal cord. Interestingly, in a zebrafish reporter gene system, these motifs were critical determinants of patterned gene expression along the rostral-caudal axis. Our findings establish a predictive model for patterned NSC gene expression, whereby domain-specific expression of LHX2 and HOX proteins act on their target motifs within commonly accessible cis-regulatory regions to specify SOX2 binding. In turn, this binding correlates strongly with these DHSs relative accessibility-a robust predictor of neighboring gene expression.
Assuntos
Cromatina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Neurais/fisiologia , Animais , Células Cultivadas , Córtex Cerebral/citologia , Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Proteínas com Homeodomínio LIM/metabolismo , Camundongos , Ligação Proteica , Fatores de Transcrição SOXB1/metabolismo , Medula Espinal/citologia , Fatores de Transcrição/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
Cell replacement therapy for diabetes mellitus requires cost-effective generation of high-quality, insulin-producing, pancreatic ß cells from pluripotent stem cells. Development of this technique has been hampered by a lack of knowledge of the molecular mechanisms underlying ß-cell differentiation. The present study identified reserpine and tetrabenazine (TBZ), both vesicular monoamine transporter 2 (VMAT2) inhibitors, as promoters of late-stage differentiation of Pdx1-positive pancreatic progenitor cells into Neurog3 (referred to henceforth as Ngn3)-positive endocrine precursors. VMAT2-controlled monoamines, such as dopamine, histamine and serotonin, negatively regulated ß-cell differentiation. Reserpine or TBZ acted additively with dibutyryl adenosine 3',5'-cyclic AMP, a cell-permeable cAMP analog, to potentiate differentiation of embryonic stem (ES) cells into ß cells that exhibited glucose-stimulated insulin secretion. When ES cell-derived ß cells were transplanted into AKITA diabetic mice, the cells reversed hyperglycemia. Our protocol provides a basis for the understanding of ß-cell differentiation and its application to a cost-effective production of functional ß cells for cell therapy.
Assuntos
Diferenciação Celular , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/metabolismo , Inibidores da Captação Adrenérgica/farmacologia , Animais , Diabetes Mellitus Experimental , Células-Tronco Embrionárias/efeitos dos fármacos , Humanos , Hiperglicemia/terapia , Camundongos , Estrutura Molecular , Reserpina/química , Reserpina/farmacologia , Tetrabenazina/química , Tetrabenazina/farmacologia , Proteínas Vesiculares de Transporte de Monoamina/antagonistas & inibidores , Proteínas Vesiculares de Transporte de Monoamina/genéticaRESUMO
BACKGROUND: Lung cancer (LC) is the leading cause of cancer-related death worldwide, including Sweden. Several studies have shown that socioeconomic status affects the risk, treatment, and survival of LC. Due to immigration after Second World War, foreign-born people constitute 12.5% of the Swedish population. We wanted to investigate if there were any differences in LC management, treatment and survival among the foreign-born Swedes (FBS) compared to the native Swedish population (NatS) in Stockholm. MATERIAL AND METHODS: A retrospective analysis of all patients diagnosed with non-small cell lung cancer (NSCLC) at the Department of Respiratory Medicine and Allergy, Karolinska University Hospital, Solna from 1 January 2003 to 31 December 2008 was made. In all, 2041 cases of LC were diagnosed, thereof 1803 with NSCLC. Of these, 211 (11.7%) were FBS. RESULTS: The mean age of NatS and FBS patients was 69.9 years, median 70 (range 26-96) and 66.0 years, median 66 (range 38-94), respectively (p < 0.001). In all, 89.8% of NatS and 90.0% of FBS were either smokers or former smokers. Adenocarcinoma was the most common subtype in both groups (NatS 54.7%, FBS 48.3%). In 140 (8.8%) of the NatS and 17 (8.1%) of the FBS the diagnosis was clinical only. There were no significant differences in stage at diagnosis, nor in performance status (PS) or different therapies between the groups. The median overall survival time for the NatS was 272 days and for FBS 328 days, again no significant difference. However, the median overall survival time for female NatS was 318 days and for female FBS 681 days (p = 0.002). CONCLUSION: FBS patients were significantly younger than NatS at diagnosis, and female FBS lived longer than female NatS, but otherwise there were no significant differences between NatS and FBS patients with LC regarding diagnosis, treatment, and survival.
Assuntos
Adenocarcinoma/mortalidade , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/terapia , Adenocarcinoma/etnologia , Adenocarcinoma/patologia , Adenocarcinoma/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/etnologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Emigrantes e Imigrantes , Feminino , Humanos , Neoplasias Pulmonares/etnologia , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Fumar , Suécia/epidemiologia , Suécia/etnologiaRESUMO
Improving the control of energy homeostasis can lower cardiovascular risk in metabolically compromised individuals. To identify new regulators of whole-body energy control, we conducted a high-throughput screen in transgenic reporter zebrafish for small molecules that modulate the expression of the fasting-inducible gluconeogenic gene pck1. We show that this in vivo strategy identified several drugs that affect gluconeogenesis in humans as well as metabolically uncharacterized compounds. Most notably, we find that the translocator protein ligands PK 11195 and Ro5-4864 are glucose-lowering agents despite a strong inductive effect on pck1 expression. We show that these drugs are activators of a fasting-like energy state and, notably, that they protect high-fat diet-induced obese mice from hepatosteatosis and glucose intolerance, two pathological manifestations of metabolic dysregulation. Thus, using a whole-organism screening strategy, this study has identified new small-molecule activators of fasting metabolism.
Assuntos
Privação de Alimentos , Animais , Animais Geneticamente Modificados , Benzodiazepinonas/farmacologia , Desenho de Fármacos , Jejum/metabolismo , Gluconeogênese , Glucose/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Isoquinolinas/farmacologia , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Transporte Proteico , Transgenes , Peixe-ZebraRESUMO
Glucose homeostasis, which is controlled by the endocrine cells of the pancreas, is disrupted in both type I and type II diabetes. Deficiency in the number of insulin-producing ß cells - a primary cause of type I diabetes and a secondary contributor of type II diabetes - leads to hyperglycemia and hence an increase in the need for insulin. Although diabetes can be controlled with insulin injections, a curative approach is needed. A potential approach to curing diabetes involves regenerating the ß-cell mass, e.g. by increasing ß-cell proliferation, survival, neogenesis or transdifferentiation. The nucleoside adenosine and its cognate nucleotide ATP have long been known to affect insulin secretion, but have more recently been shown to increase ß-cell proliferation during homeostatic control and regeneration of the ß-cell mass. Adenosine is also known to have anti-inflammatory properties, and agonism of adenosine receptors can promote the survival of ß-cells in an inflammatory microenvironment. In this review, both intracellular and extracellular mechanisms of adenosine and ATP are discussed in terms of their established and putative effects on ß-cell regeneration.
Assuntos
Adenosina/metabolismo , Diabetes Mellitus/patologia , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Regeneração/fisiologia , Animais , Diabetes Mellitus/metabolismo , Humanos , Transdução de SinaisRESUMO
The zebrafish has become an outstanding model for studying organ development and tissue regeneration, which is prominently leveraged for studies of pancreatic development, insulin-producing ß-cells, and diabetes. Although studied for more than two decades, many aspects remain elusive and it has only recently been possible to investigate these due to technical advances in transcriptomics, chemical-genetics, genome editing, drug screening, and in vivo imaging. Here, we review recent findings on zebrafish pancreas development, ß-cell regeneration, and how zebrafish can be used to provide novel insights into gene functions, disease mechanisms, and therapeutic targets in diabetes, inspiring further use of zebrafish for the development of novel therapies for diabetes.
RESUMO
Insulin binds the insulin receptor (IR) and regulates anabolic processes in target tissues. Impaired IR signalling is associated with multiple diseases, including diabetes, cancer and neurodegenerative disorders. IRs have been reported to form nanoclusters at the cell membrane in several cell types, even in the absence of insulin binding. Here we exploit the nanoscale spatial organization of the IR to achieve controlled multivalent receptor activation. To control insulin nanoscale spatial organization and valency, we developed rod-like insulin-DNA origami nanostructures carrying different numbers of insulin molecules with defined spacings. Increasing the insulin valency per nanostructure markedly extended the residence time of insulin-DNA origami nanostructures at the receptors. Both insulin valency and spacing affected the levels of IR activation in adipocytes. Moreover, the multivalent insulin design associated with the highest levels of IR activation also induced insulin-mediated transcriptional responses more effectively than the corresponding monovalent insulin nanostructures. In an in vivo zebrafish model of diabetes, treatment with multivalent-but not monovalent-insulin nanostructures elicited a reduction in glucose levels. Our results show that the control of insulin multivalency and spatial organization with nanoscale precision modulates the IR responses, independent of the insulin concentration. Therefore, we propose insulin nanoscale organization as a design parameter in developing new insulin therapies.
Assuntos
DNA , Nanoestruturas , Receptor de Insulina , Animais , Diabetes Mellitus/tratamento farmacológico , DNA/química , Insulina , Nanoestruturas/química , Receptor de Insulina/efeitos dos fármacos , Receptor de Insulina/metabolismo , Peixe-ZebraRESUMO
Regeneration of insulin-producing ß-cells is an alternative avenue to manage diabetes, and it is crucial to unravel this process in vivo during physiological responses to the lack of ß-cells. Here, we aimed to characterize how hepatocytes can contribute to ß-cell regeneration, either directly or indirectly via secreted proteins or metabolites, in a zebrafish model of ß-cell loss. Using lineage tracing, we show that hepatocytes do not directly convert into ß-cells even under extreme ß-cell ablation conditions. A transcriptomic analysis of isolated hepatocytes after ß-cell ablation displayed altered lipid- and glucose-related processes. Based on the transcriptomics, we performed a genetic screen that uncovers a potential role of the molybdenum cofactor (Moco) biosynthetic pathway in ß-cell regeneration and glucose metabolism in zebrafish. Consistently, molybdenum cofactor synthesis 2 (Mocs2) haploinsufficiency in mice indicated dysregulated glucose metabolism and liver function. Together, our study sheds light on the liver-pancreas crosstalk and suggests that the molybdenum cofactor biosynthesis pathway should be further studied in relation to glucose metabolism and diabetes.
Assuntos
Coenzimas , Glucose , Hepatócitos , Células Secretoras de Insulina , Fígado , Metaloproteínas , Cofatores de Molibdênio , Pteridinas , Peixe-Zebra , Animais , Células Secretoras de Insulina/metabolismo , Pteridinas/metabolismo , Coenzimas/metabolismo , Camundongos , Fígado/metabolismo , Fígado/citologia , Metaloproteínas/metabolismo , Metaloproteínas/genética , Hepatócitos/metabolismo , Glucose/metabolismo , Regeneração/genética , Pâncreas/metabolismo , Pâncreas/citologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The development of craniofacial skeletal structures is fascinatingly complex and elucidation of the underlying mechanisms will not only provide novel scientific insights, but also help develop more effective clinical approaches to the treatment and/or prevention of the numerous congenital craniofacial malformations. To this end, we performed a genome-wide analysis of RNA transcription from non-coding regulatory elements by CAGE-sequencing of the facial mesenchyme of human embryos and cross-checked the active enhancers thus identified against genes, identified by GWAS for the normal range human facial appearance. Among the identified active cis-enhancers, several belonged to the components of the PI3/AKT/mTORC1/autophagy pathway. To assess the functional role of this pathway, we manipulated it both genetically and pharmacologically in mice and zebrafish. These experiments revealed that mTORC1 signaling modulates craniofacial shaping at the stage of skeletal mesenchymal condensations, with subsequent fine-tuning during clonal intercalation. This ability of mTORC1 pathway to modulate facial shaping, along with its evolutionary conservation and ability to sense external stimuli, in particular dietary amino acids, indicate that the mTORC1 pathway may play a role in facial phenotypic plasticity. Indeed, the level of protein in the diet of pregnant female mice influenced the activity of mTORC1 in fetal craniofacial structures and altered the size of skeletogenic clones, thus exerting an impact on the local geometry and craniofacial shaping. Overall, our findings indicate that the mTORC1 signaling pathway is involved in the effect of environmental conditions on the shaping of craniofacial structures.
Assuntos
Transdução de Sinais , Peixe-Zebra , Gravidez , Camundongos , Animais , Feminino , Humanos , Proteínas , Alvo Mecanístico do Complexo 1 de Rapamicina , DietaRESUMO
The oxidative phosphorylation system1 in mammalian mitochondria plays a key role in transducing energy from ingested nutrients2. Mitochondrial metabolism is dynamic and can be reprogrammed to support both catabolic and anabolic reactions, depending on physiological demands or disease states. Rewiring of mitochondrial metabolism is intricately linked to metabolic diseases and promotes tumour growth3-5. Here, we demonstrate that oral treatment with an inhibitor of mitochondrial transcription (IMT)6 shifts whole-animal metabolism towards fatty acid oxidation, which, in turn, leads to rapid normalization of body weight, reversal of hepatosteatosis and restoration of normal glucose tolerance in male mice on a high-fat diet. Paradoxically, the IMT treatment causes a severe reduction of oxidative phosphorylation capacity concomitant with marked upregulation of fatty acid oxidation in the liver, as determined by proteomics and metabolomics analyses. The IMT treatment leads to a marked reduction of complex I, the main dehydrogenase feeding electrons into the ubiquinone (Q) pool, whereas the levels of electron transfer flavoprotein dehydrogenase and other dehydrogenases connected to the Q pool are increased. This rewiring of metabolism caused by reduced mtDNA expression in the liver provides a principle for drug treatment of obesity and obesity-related pathology.
Assuntos
DNA Mitocondrial , Dieta Hiperlipídica , Obesidade , Transcrição Gênica , Animais , Obesidade/metabolismo , Obesidade/etiologia , Camundongos , DNA Mitocondrial/metabolismo , Masculino , Fígado Gorduroso/metabolismo , Fígado Gorduroso/etiologia , Fosforilação Oxidativa , Fígado/metabolismo , Ácidos Graxos/metabolismo , Camundongos Endogâmicos C57BL , OxirreduçãoRESUMO
Bmp signaling has been shown to regulate early aspects of pancreas development, but its role in endocrine, and especially beta-cell, differentiation remains unclear. Taking advantage of the ability in zebrafish embryos to cell-autonomously modulate Bmp signaling in single cells, we examined how Bmp signaling regulates the ability of individual endodermal cells to differentiate into beta-cells. We find that specific temporal windows of Bmp signaling prevent beta-cell differentiation. Thus, future dorsal bud-derived beta-cells are sensitive to Bmp signaling specifically during gastrulation and early somitogenesis stages. In contrast, ventral pancreatic cells, which require an early Bmp signal to form, do not produce beta-cells when exposed to Bmp signaling at 50 hpf, a stage when the ventral bud-derived extrapancreatic duct is the main source of new endocrine cells. Importantly, inhibiting Bmp signaling within endodermal cells via genetic means increased the number of beta-cells, at early and late stages. Moreover, inhibition of Bmp signaling in the late stage embryo using dorsomorphin, a chemical inhibitor of Bmp receptors, significantly increased beta-cell neogenesis near the extrapancreatic duct, demonstrating the feasibility of pharmacological approaches to increase beta-cell numbers. Our in vivo single-cell analyses show that whereas Bmp signaling is necessary initially for formation of the ventral pancreas, differentiating endodermal cells need to be protected from exposure to Bmps during specific stages to permit beta-cell differentiation. These results provide important unique insight into the intercellular signaling environment necessary for in vivo and in vitro generation of beta-cells.
Assuntos
Receptores de Ativinas Tipo I/fisiologia , Proteínas Morfogenéticas Ósseas/fisiologia , Indução Embrionária/fisiologia , Ilhotas Pancreáticas/citologia , Transdução de Sinais/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Animais , Sequência de Bases , Primers do DNA , Hibridização In SituRESUMO
Here, we devised a cloning-free 3' knock-in strategy for zebrafish using PCR amplified dsDNA donors that avoids disrupting the targeted genes. The dsDNA donors carry genetic cassettes coding for fluorescent proteins and Cre recombinase in frame with the endogenous gene but separated from it by self-cleavable peptides. Primers with 5' AmC6 end-protections generated PCR amplicons with increased integration efficiency that were coinjected with preassembled Cas9/gRNA ribonucleoprotein complexes for early integration. We targeted four genetic loci (krt92, nkx6.1, krt4, and id2a) and generated 10 knock-in lines, which function as reporters for the endogenous gene expression. The knocked-in iCre or CreERT2 lines were used for lineage tracing, which suggested that nkx6.1 + cells are multipotent pancreatic progenitors that gradually restrict to the bipotent duct, whereas id2a + cells are multipotent in both liver and pancreas and gradually restrict to ductal cells. In addition, the hepatic id2a + duct show progenitor properties upon extreme hepatocyte loss. Thus, we present an efficient and straightforward knock-in technique with widespread use for cellular labelling and lineage tracing.