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1.
Nature ; 590(7845): 326-331, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33505018

RESUMEN

Resistance to insulin and insulin-like growth factor 1 (IGF1) in pancreatic ß-cells causes overt diabetes in mice; thus, therapies that sensitize ß-cells to insulin may protect patients with diabetes against ß-cell failure1-3. Here we identify an inhibitor of insulin receptor (INSR) and IGF1 receptor (IGF1R) signalling in mouse ß-cells, which we name the insulin inhibitory receptor (inceptor; encoded by the gene Iir). Inceptor contains an extracellular cysteine-rich domain with similarities to INSR and IGF1R4, and a mannose 6-phosphate receptor domain that is also found in the IGF2 receptor (IGF2R)5. Knockout mice that lack inceptor (Iir-/-) exhibit signs of hyperinsulinaemia and hypoglycaemia, and die within a few hours of birth. Molecular and cellular analyses of embryonic and postnatal pancreases from Iir-/- mice showed an increase in the activation of INSR-IGF1R in Iir-/- pancreatic tissue, resulting in an increase in the proliferation and mass of ß-cells. Similarly, inducible ß-cell-specific Iir-/- knockout in adult mice and in ex vivo islets led to an increase in the activation of INSR-IGF1R and increased proliferation of ß-cells, resulting in improved glucose tolerance in vivo. Mechanistically, inceptor interacts with INSR-IGF1R to facilitate clathrin-mediated endocytosis for receptor desensitization. Blocking this physical interaction using monoclonal antibodies against the extracellular domain of inceptor resulted in the retention of inceptor and INSR at the plasma membrane to sustain the activation of INSR-IGF1R in ß-cells. Together, our findings show that inceptor shields insulin-producing ß-cells from constitutive pathway activation, and identify inceptor as a potential molecular target for INSR-IGF1R sensitization and diabetes therapy.


Asunto(s)
Glucemia/metabolismo , Antagonistas de Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas de Neoplasias/metabolismo , Transducción de Señal , Animales , Glucemia/análisis , Línea Celular , Proliferación Celular/efectos de los fármacos , Tamaño de la Célula , Clatrina/metabolismo , Células Endocrinas/metabolismo , Endocitosis , Retículo Endoplásmico/metabolismo , Prueba de Tolerancia a la Glucosa , Aparato de Golgi/metabolismo , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Lisosomas/metabolismo , Masculino , Proteínas de la Membrana , Ratones , Proteínas de Neoplasias/química , Receptor de Insulina/metabolismo , Transducción de Señal/efectos de los fármacos , Tamoxifeno/farmacología
2.
Development ; 146(12)2019 06 17.
Artículo en Inglés | MEDLINE | ID: mdl-31160421

RESUMEN

Deciphering mechanisms of endocrine cell induction, specification and lineage allocation in vivo will provide valuable insights into how the islets of Langerhans are generated. Currently, it is ill defined how endocrine progenitors segregate into different endocrine subtypes during development. Here, we generated a novel neurogenin 3 (Ngn3)-Venus fusion (NVF) reporter mouse line, that closely mirrors the transient endogenous Ngn3 protein expression. To define an in vivo roadmap of endocrinogenesis, we performed single cell RNA sequencing of 36,351 pancreatic epithelial and NVF+ cells during secondary transition. This allowed Ngn3low endocrine progenitors, Ngn3high endocrine precursors, Fev+ endocrine lineage and hormone+ endocrine subtypes to be distinguished and time-resolved, and molecular programs during the step-wise lineage restriction steps to be delineated. Strikingly, we identified 58 novel signature genes that show the same transient expression dynamics as Ngn3 in the 7260 profiled Ngn3-expressing cells. The differential expression of these genes in endocrine precursors associated with their cell-fate allocation towards distinct endocrine cell types. Thus, the generation of an accurately regulated NVF reporter allowed us to temporally resolve endocrine lineage development to provide a fine-grained single cell molecular profile of endocrinogenesis in vivo.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas del Tejido Nervioso/genética , Páncreas/embriología , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Animales , Diferenciación Celular/genética , Linaje de la Célula , Células Endocrinas/citología , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Células Secretoras de Insulina/citología , Ratones , Regeneración , Transducción de Señal , Células Madre/citología , Proteínas Wnt/metabolismo
4.
Int J Mol Sci ; 22(22)2021 Nov 20.
Artículo en Inglés | MEDLINE | ID: mdl-34830411

RESUMEN

Synaptotagmin-13 (Syt13) is an atypical member of the vesicle trafficking synaptotagmin protein family. The expression pattern and the biological function of this Ca2+-independent protein are not well resolved. Here, we have generated a novel Syt13-Venus fusion (Syt13-VF) fluorescence reporter allele to track and isolate tissues and cells expressing Syt13 protein. The reporter allele is regulated by endogenous cis-regulatory elements of Syt13 and the fusion protein follows an identical expression pattern of the endogenous Syt13 protein. The homozygous reporter mice are viable and fertile. We identify the expression of the Syt13-VF reporter in different regions of the brain with high expression in tyrosine hydroxylase (TH)-expressing and oxytocin-producing neuroendocrine cells. Moreover, Syt13-VF is highly restricted to all enteroendocrine cells in the adult intestine that can be traced in live imaging. Finally, Syt13-VF protein is expressed in the pancreatic endocrine lineage, allowing their specific isolation by flow sorting. These findings demonstrate high expression levels of Syt13 in the endocrine lineages in three major organs harboring these secretory cells. Collectively, the Syt13-VF reporter mouse line provides a unique and reliable tool to dissect the spatio-temporal expression pattern of Syt13 and enables isolation of Syt13-expressing cells that will aid in deciphering the molecular functions of this protein in the neuroendocrine system.


Asunto(s)
Encéfalo/metabolismo , Intestinos/metabolismo , Páncreas/metabolismo , Sinaptotagminas/genética , Animales , Encéfalo/patología , Línea Celular Tumoral , Linaje de la Célula/genética , Movimiento Celular/genética , Regulación de la Expresión Génica/genética , Humanos , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Ratones , Sistemas Neurosecretores/metabolismo , Sistemas Neurosecretores/patología , Oxitocina/genética , Sinaptotagminas/metabolismo , Tirosina 3-Monooxigenasa/genética
5.
Development ; 144(16): 2873-2888, 2017 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-28811309

RESUMEN

The pancreas is an endoderm-derived glandular organ that participates in the regulation of systemic glucose metabolism and food digestion through the function of its endocrine and exocrine compartments, respectively. While intensive research has explored the signaling pathways and transcriptional programs that govern pancreas development, much remains to be discovered regarding the cellular processes that orchestrate pancreas morphogenesis. Here, we discuss the developmental mechanisms and principles that are known to underlie pancreas development, from induction and lineage formation to morphogenesis and organogenesis. Elucidating such principles will help to identify novel candidate disease genes and unravel the pathogenesis of pancreas-related diseases, such as diabetes, pancreatitis and cancer.


Asunto(s)
Páncreas/embriología , Animales , Endodermo/embriología , Endodermo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Páncreas/metabolismo , Transducción de Señal
6.
Int J Mol Sci ; 20(21)2019 Oct 30.
Artículo en Inglés | MEDLINE | ID: mdl-31671683

RESUMEN

The exponential increase of patients with diabetes mellitus urges for novel therapeutic strategies to reduce the socioeconomic burden of this disease. The loss or dysfunction of insulin-producing ß-cells, in patients with type 1 and type 2 diabetes respectively, put these cells at the center of the disease initiation and progression. Therefore, major efforts have been taken to restore the ß-cell mass by cell-replacement or regeneration approaches. Implementing novel therapies requires deciphering the developmental mechanisms that generate ß-cells and determine the acquisition of their physiological phenotype. In this review, we summarize the current understanding of the mechanisms that coordinate the postnatal maturation of ß-cells and define their functional identity. Furthermore, we discuss different routes by which ß-cells lose their features and functionality in type 1 and 2 diabetic conditions. We then focus on potential mechanisms to restore the functionality of those ß-cell populations that have lost their functional phenotype. Finally, we discuss the recent progress and remaining challenges facing the generation of functional mature ß-cells from stem cells for cell-replacement therapy for diabetes treatment.


Asunto(s)
Diabetes Mellitus/terapia , Células Secretoras de Insulina/citología , Diferenciación Celular , Transdiferenciación Celular , Diabetes Mellitus/metabolismo , Progresión de la Enfermedad , Humanos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/trasplante , Fenotipo , Transducción de Señal
7.
Dev Biol ; 429(1): 56-70, 2017 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-28733161

RESUMEN

The ventral mesencephalic neural precursor cells (vmNPCs) that give rise to dopaminergic (DA) neurons have been identified by the expression of distinct genes (e.g., Lmx1a, Foxa2, Msx1/2). However, the commitment of these NPCs to the mesencephalic DA neuronal fate has not been functionally determined. Evaluation of the plasticity of vmNPCs suggests that their commitment occurs after E10.5. Here we show that E9.5 vmNPCs implanted in an ectopic area of E10.5 mesencephalic explants, retained their specification marker Lmx1a and efficiently differentiated into neurons but did not express the gene encoding tyrosine hydroxylase (Th), the limiting enzyme for dopamine synthesis. A proportion of E10.5-E11.5 implanted vmNPCs behaved as committed, deriving into Th+ neurons in ectopic sites. Interestingly, implanted cells from E12.5 embryos were unable to give rise to a significant number of Th+ neurons. Concomitantly, differentiation assays in culture and in mesencephalic explants treated with Fgf2+LIF detected vmNPCs with astrogenic potential since E11.5. Despite this, a full suspension of E12.5 vmNPCs give rise to DA neurons in a similar proportion as those of E10.5 when they were transplanted into adult brain, but astrocytes were only detected with the former population. These data suggest that the subventricular postmitotic progenitors present in E12.5 ventral mesencephalon are unable to implant in embryonic explants and are the source of DA neurons in the transplanted adult brain. Based on our findings we propose that during DA differentiation committed vmNPCs emerge at E10.5 and they exhaust their neurogenic capacity with the rise of NPCs with astrogenic potential.


Asunto(s)
Diferenciación Celular , Neuronas Dopaminérgicas/citología , Mesencéfalo/citología , Células-Madre Neurales/citología , Neurogénesis , Animales , Astrocitos/citología , Linaje de la Célula , Proliferación Celular , Supervivencia Celular , Neuronas Dopaminérgicas/metabolismo , Embrión de Mamíferos/citología , Femenino , Proteínas Hedgehog/metabolismo , Proteínas con Homeodominio LIM/metabolismo , Masculino , Ratones Transgénicos , Modelos Biológicos , Células-Madre Neurales/metabolismo , Ratas Wistar , Nicho de Células Madre , Factores de Transcripción/metabolismo
8.
Mol Metab ; 79: 101853, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38103636

RESUMEN

OBJECTIVE: The consequences of mutations in genes associated with monogenic forms of diabetes on human pancreas development cannot be studied in a time-resolved fashion in vivo. More specifically, if recessive mutations in the insulin gene influence human pancreatic endocrine lineage formation is still an unresolved question. METHODS: To model the extremely reduced insulin levels in patients with recessive insulin gene mutations, we generated a novel knock-in H2B-Cherry reporter human induced pluripotent stem cell (iPSC) line expressing no insulin upon differentiation to stem cell-derived (SC-) ß cells in vitro. Differentiation of iPSCs into the pancreatic and endocrine lineage, combined with immunostaining, Western blotting and proteomics analysis phenotypically characterized the insulin gene deficiency in SC-islets. Furthermore, we leveraged FACS analysis and confocal microscopy to explore the impact of insulin shortage on human endocrine cell induction, composition, differentiation and proliferation. RESULTS: Interestingly, insulin-deficient SC-islets exhibited low insulin receptor (IR) signaling when stimulated with glucose but displayed increased IR sensitivity upon treatment with exogenous insulin. Furthermore, insulin shortage did not alter neurogenin-3 (NGN3)-mediated endocrine lineage induction. Nevertheless, lack of insulin skewed the SC-islet cell composition with an increased number in SC-ß cell formation at the expense of SC-α cells. Finally, insulin deficiency reduced the rate of SC-ß cell proliferation but had no impact on the expansion of SC-α cells. CONCLUSIONS: Using iPSC disease modelling, we provide first evidence of insulin function in human pancreatic endocrine lineage formation. These findings help to better understand the phenotypic impact of recessive insulin gene mutations during pancreas development and shed light on insulin gene function beside its physiological role in blood glucose regulation.


Asunto(s)
Células Endocrinas , Células Madre Pluripotentes Inducidas , Humanos , Insulina/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Diferenciación Celular/genética , Páncreas/metabolismo , Insulina Regular Humana/metabolismo , Células Endocrinas/metabolismo
9.
Nat Metab ; 6(3): 448-457, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38418586

RESUMEN

Insulin resistance is an early complication of diet-induced obesity (DIO)1, potentially leading to hyperglycaemia and hyperinsulinaemia, accompanied by adaptive ß cell hypertrophy and development of type 2 diabetes2. Insulin not only signals via the insulin receptor (INSR), but also promotes ß cell survival, growth and function via the insulin-like growth factor 1 receptor (IGF1R)3-6. We recently identified the insulin inhibitory receptor (inceptor) as the key mediator of IGF1R and INSR desensitization7. But, although ß cell-specific loss of inceptor improves ß cell function in lean mice7, it warrants clarification whether inceptor signal inhibition also improves glycaemia under conditions of obesity. We assessed the glucometabolic effects of targeted inceptor deletion in either the brain or the pancreatic ß cells under conditions of DIO in male mice. In the present study, we show that global and neuronal deletion of inceptor, as well as its adult-onset deletion in the ß cells, improves glucose homeostasis by enhancing ß cell health and function. Moreover, we demonstrate that inceptor-mediated improvement in glucose control does not depend on inceptor function in agouti-related protein-expressing or pro-opiomelanocortin neurons. Our data demonstrate that inceptor inhibition improves glucose homeostasis in mice with DIO, hence corroborating that inceptor is a crucial regulator of INSR and IGF1R signalling.


Asunto(s)
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Ratones , Masculino , Animales , Células Secretoras de Insulina/metabolismo , Glucosa/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Obesidad/genética , Obesidad/metabolismo , Dieta , Insulina/metabolismo , Homeostasis , Neuronas/metabolismo
10.
Mol Metab ; 83: 101915, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38492844

RESUMEN

OBJECTIVE: The glucose-dependent insulinotropic polypeptide (GIP) decreases body weight via central GIP receptor (GIPR) signaling, but the underlying mechanisms remain largely unknown. Here, we assessed whether GIP regulates body weight and glucose control via GIPR signaling in cells that express the leptin receptor (Lepr). METHODS: Hypothalamic, hindbrain, and pancreatic co-expression of Gipr and Lepr was assessed using single cell RNAseq analysis. Mice with deletion of Gipr in Lepr cells were generated and metabolically characterized for alterations in diet-induced obesity (DIO), glucose control and leptin sensitivity. Long-acting single- and dual-agonists at GIPR and GLP-1R were further used to assess drug effects on energy and glucose metabolism in DIO wildtype (WT) and Lepr-Gipr knock-out (KO) mice. RESULTS: Gipr and Lepr show strong co-expression in the pancreas, but not in the hypothalamus and hindbrain. DIO Lepr-Gipr KO mice are indistinguishable from WT controls related to body weight, food intake and diet-induced leptin resistance. Acyl-GIP and the GIPR:GLP-1R co-agonist MAR709 remain fully efficacious to decrease body weight and food intake in DIO Lepr-Gipr KO mice. Consistent with the demonstration that Gipr and Lepr highly co-localize in the endocrine pancreas, including the ß-cells, we find the superior glycemic effect of GIPR:GLP-1R co-agonism over single GLP-1R agonism to vanish in Lepr-Gipr KO mice. CONCLUSIONS: GIPR signaling in cells/neurons that express the leptin receptor is not implicated in the control of body weight or food intake, but is of crucial importance for the superior glycemic effects of GIPR:GLP-1R co-agonism relative to single GLP-1R agonism.


Asunto(s)
Peso Corporal , Ingestión de Alimentos , Polipéptido Inhibidor Gástrico , Ratones Noqueados , Obesidad , Receptores de la Hormona Gastrointestinal , Receptores de Leptina , Animales , Masculino , Ratones , Polipéptido Inhibidor Gástrico/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón/genética , Glucosa/metabolismo , Leptina/metabolismo , Ratones Endogámicos C57BL , Obesidad/metabolismo , Receptores de la Hormona Gastrointestinal/metabolismo , Receptores de la Hormona Gastrointestinal/genética , Receptores de Leptina/metabolismo , Receptores de Leptina/genética , Transducción de Señal
11.
Nat Metab ; 5(9): 1615-1637, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37697055

RESUMEN

Although multiple pancreatic islet single-cell RNA-sequencing (scRNA-seq) datasets have been generated, a consensus on pancreatic cell states in development, homeostasis and diabetes as well as the value of preclinical animal models is missing. Here, we present an scRNA-seq cross-condition mouse islet atlas (MIA), a curated resource for interactive exploration and computational querying. We integrate over 300,000 cells from nine scRNA-seq datasets consisting of 56 samples, varying in age, sex and diabetes models, including an autoimmune type 1 diabetes model (NOD), a glucotoxicity/lipotoxicity type 2 diabetes model (db/db) and a chemical streptozotocin ß-cell ablation model. The ß-cell landscape of MIA reveals new cell states during disease progression and cross-publication differences between previously suggested marker genes. We show that ß-cells in the streptozotocin model transcriptionally correlate with those in human type 2 diabetes and mouse db/db models, but are less similar to human type 1 diabetes and mouse NOD ß-cells. We also report pathways that are shared between ß-cells in immature, aged and diabetes models. MIA enables a comprehensive analysis of ß-cell responses to different stressors, providing a roadmap for the understanding of ß-cell plasticity, compensation and demise.


Asunto(s)
Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 2 , Humanos , Animales , Ratones , Anciano , Ratones Endogámicos NOD , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 2/genética , Estreptozocina , Modelos Animales de Enfermedad
12.
Front Endocrinol (Lausanne) ; 14: 1286590, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37955006

RESUMEN

Introduction: The molecular programs regulating human pancreatic endocrine cell induction and fate allocation are not well deciphered. Here, we investigated the spatiotemporal expression pattern and the function of the neurogenic differentiation factor 2 (NEUROD2) during human endocrinogenesis. Methods: Using Crispr-Cas9 gene editing, we generated a reporter knock-in transcription factor (TF) knock-out human inducible pluripotent stem cell (iPSC) line in which the open reading frame of both NEUROD2 alleles are replaced by a nuclear histone 2B-Venus reporter (NEUROD2nVenus/nVenus). Results: We identified a transient expression of NEUROD2 mRNA and its nuclear Venus reporter activity at the stage of human endocrine progenitor formation in an iPSC differentiation model. This expression profile is similar to what was previously reported in mice, uncovering an evolutionarily conserved gene expression pattern of NEUROD2 during endocrinogenesis. In vitro differentiation of the generated homozygous NEUROD2nVenus/nVenus iPSC line towards human endocrine lineages uncovered no significant impact upon the loss of NEUROD2 on endocrine cell induction. Moreover, analysis of endocrine cell specification revealed no striking changes in the generation of insulin-producing b cells and glucagon-secreting a cells upon lack of NEUROD2. Discussion: Overall, our results suggest that NEUROD2 is expendable for human b cell formation in vitro.


Asunto(s)
Células Secretoras de Insulina , Neuropéptidos , Humanos , Animales , Ratones , Células Secretoras de Insulina/metabolismo , Factores de Transcripción/metabolismo , Diferenciación Celular/genética , Línea Celular , Páncreas , Neuropéptidos/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo
13.
Nat Metab ; 5(12): 2075-2085, 2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-37946085

RESUMEN

The development of single-molecule co-agonists for the glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) is considered a breakthrough in the treatment of obesity and type 2 diabetes. But although GIPR-GLP-1R co-agonism decreases body weight with superior efficacy relative to GLP-1R agonism alone in preclinical1-3 and clinical studies4,5, the role of GIP in regulating energy metabolism remains enigmatic. Increasing evidence suggests that long-acting GIPR agonists act in the brain to decrease body weight through the inhibition of food intake3,6-8; however, the mechanisms and neuronal populations through which GIP affects metabolism remain to be identified. Here, we report that long-acting GIPR agonists and GIPR-GLP-1R co-agonists decrease body weight and food intake via inhibitory GABAergic neurons. We show that acyl-GIP decreases body weight and food intake in male diet-induced obese wild-type mice, but not in mice with deletion of Gipr in Vgat(also known as Slc32a1)-expressing GABAergic neurons (Vgat-Gipr knockout). Whereas the GIPR-GLP-1R co-agonist MAR709 leads, in male diet-induced obese wild-type mice, to greater weight loss and further inhibition of food intake relative to a pharmacokinetically matched acyl-GLP-1 control, this superiority over GLP-1 vanishes in Vgat-Gipr knockout mice. Our data demonstrate that long-acting GIPR agonists crucially depend on GIPR signaling in inhibitory GABAergic neurons to decrease body weight and food intake.


Asunto(s)
Diabetes Mellitus Tipo 2 , Masculino , Ratones , Animales , Diabetes Mellitus Tipo 2/metabolismo , Polipéptido Inhibidor Gástrico/metabolismo , Obesidad/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , Receptores Acoplados a Proteínas G , Glucosa , Neuronas GABAérgicas/metabolismo , Ingestión de Alimentos
14.
Nat Commun ; 13(1): 4540, 2022 08 04.
Artículo en Inglés | MEDLINE | ID: mdl-35927244

RESUMEN

During pancreas development endocrine cells leave the ductal epithelium to form the islets of Langerhans, but the morphogenetic mechanisms are incompletely understood. Here, we identify the Ca2+-independent atypical Synaptotagmin-13 (Syt13) as a key regulator of endocrine cell egression and islet formation. We detect specific upregulation of the Syt13 gene and encoded protein in endocrine precursors and the respective lineage during islet formation. The Syt13 protein is localized to the apical membrane of endocrine precursors and to the front domain of egressing endocrine cells, marking a previously unidentified apical-basal to front-rear repolarization during endocrine precursor cell egression. Knockout of Syt13 impairs endocrine cell egression and skews the α-to-ß-cell ratio. Mechanistically, Syt13 is a vesicle trafficking protein, transported via the microtubule cytoskeleton, and interacts with phosphatidylinositol phospholipids for polarized localization. By internalizing a subset of plasma membrane proteins at the front domain, including α6ß4 integrins, Syt13 modulates cell-matrix adhesion and allows efficient endocrine cell egression. Altogether, these findings uncover an unexpected role for Syt13 as a morphogenetic driver of endocrinogenesis and islet formation.


Asunto(s)
Células Endocrinas , Islotes Pancreáticos , Integrinas , Morfogénesis , Páncreas , Sinaptotagminas/genética
15.
Nat Metab ; 4(8): 1071-1083, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35995995

RESUMEN

Dual agonists activating the peroxisome proliferator-activated receptors alpha and gamma (PPARɑ/ɣ) have beneficial effects on glucose and lipid metabolism in patients with type 2 diabetes, but their development was discontinued due to potential adverse effects. Here we report the design and preclinical evaluation of a molecule that covalently links the PPARɑ/ɣ dual-agonist tesaglitazar to a GLP-1 receptor agonist (GLP-1RA) to allow for GLP-1R-dependent cellular delivery of tesaglitazar. GLP-1RA/tesaglitazar does not differ from the pharmacokinetically matched GLP-1RA in GLP-1R signalling, but shows GLP-1R-dependent PPARɣ-retinoic acid receptor heterodimerization and enhanced improvements of body weight, food intake and glucose metabolism relative to the GLP-1RA or tesaglitazar alone in obese male mice. The conjugate fails to affect body weight and glucose metabolism in GLP-1R knockout mice and shows preserved effects in obese mice at subthreshold doses for the GLP-1RA and tesaglitazar. Liquid chromatography-mass spectrometry-based proteomics identified PPAR regulated proteins in the hypothalamus that are acutely upregulated by GLP-1RA/tesaglitazar. Our data show that GLP-1RA/tesaglitazar improves glucose control with superior efficacy to the GLP-1RA or tesaglitazar alone and suggest that this conjugate might hold therapeutic value to acutely treat hyperglycaemia and insulin resistance.


Asunto(s)
Diabetes Mellitus Tipo 2 , PPAR alfa , Alcanosulfonatos , Animales , Peso Corporal , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Péptido 1 Similar al Glucagón/uso terapéutico , Receptor del Péptido 1 Similar al Glucagón , Glucosa , Masculino , Ratones , Obesidad/tratamiento farmacológico , Obesidad/metabolismo , PPAR alfa/agonistas , PPAR alfa/uso terapéutico , Fenilpropionatos
16.
Mol Metab ; 54: 101330, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34500108

RESUMEN

OBJECTIVE: The effectiveness of bariatric surgery in restoring ß-cell function has been described in type-2 diabetes (T2D) patients and animal models for years, whereas the mechanistic underpinnings are largely unknown. The possibility of vertical sleeve gastrectomy (VSG) to rescue far-progressed, clinically-relevant T2D and to promote ß-cell recovery has not been investigated on a single-cell level. Nevertheless, characterization of the heterogeneity and functional states of ß-cells after VSG is a fundamental step to understand mechanisms of glycaemic recovery and to ultimately develop alternative, less-invasive therapies. METHODS: We performed VSG in late-stage diabetic db/db mice and analyzed the islet transcriptome using single-cell RNA sequencing (scRNA-seq). Immunohistochemical analyses and quantification of ß-cell area and proliferation complement our findings from scRNA-seq. RESULTS: We report that VSG was superior to calorie restriction in late-stage T2D and rapidly restored normoglycaemia in morbidly obese and overt diabetic db/db mice. Single-cell profiling of islets of Langerhans showed that VSG induced distinct, intrinsic changes in the ß-cell transcriptome, but not in that of α-, δ-, and PP-cells. VSG triggered fast ß-cell redifferentiation and functional improvement within only two weeks of intervention, which is not seen upon calorie restriction. Furthermore, VSG expanded ß-cell area by means of redifferentiation and by creating a proliferation competent ß-cell state. CONCLUSION: Collectively, our study reveals the superiority of VSG in the remission of far-progressed T2D and presents paths of ß-cell regeneration and molecular pathways underlying the glycaemic benefits of VSG.


Asunto(s)
Diabetes Mellitus Tipo 2/cirugía , Gastrectomía , Células Secretoras de Insulina/patología , Animales , Masculino , Ratones , Ratones Obesos , Ratones Transgénicos
17.
Mol Metab ; 49: 101188, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-33582383

RESUMEN

OBJECTIVE: Islets of Langerhans contain heterogeneous populations of insulin-producing ß-cells. Surface markers and respective antibodies for isolation, tracking, and analysis are urgently needed to study ß-cell heterogeneity and explore the mechanisms to harness the regenerative potential of immature ß-cells. METHODS: We performed single-cell mRNA profiling of early postnatal mouse islets and re-analyzed several single-cell mRNA sequencing datasets from mouse and human pancreas and islets. We used mouse primary islets, iPSC-derived endocrine cells, Min6 insulinoma, and human EndoC-ßH1 ß-cell lines and performed FAC sorting, Western blotting, and imaging to support and complement the findings from the data analyses. RESULTS: We found that all endocrine cell types expressed the cluster of differentiation 81 (CD81) during pancreas development, but the expression levels of this protein were gradually reduced in ß-cells during postnatal maturation. Single-cell gene expression profiling and high-resolution imaging revealed an immature signature of ß-cells expressing high levels of CD81 (CD81high) compared to a more mature population expressing no or low levels of this protein (CD81low/-). Analysis of ß-cells from different diabetic mouse models and in vitro ß-cell stress assays indicated an upregulation of CD81 expression levels in stressed and dedifferentiated ß-cells. Similarly, CD81 was upregulated and marked stressed human ß-cells in vitro. CONCLUSIONS: We identified CD81 as a novel surface marker that labels immature, stressed, and dedifferentiated ß-cells in the adult mouse and human islets. This novel surface marker will allow us to better study ß-cell heterogeneity in healthy subjects and diabetes progression.


Asunto(s)
Diferenciación Celular , Células Secretoras de Insulina/metabolismo , Tetraspanina 28/genética , Tetraspanina 28/metabolismo , Animales , Línea Celular , Diabetes Mellitus/metabolismo , Femenino , Expresión Génica , Perfilación de la Expresión Génica/métodos , Heterogeneidad Genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Ratones , Páncreas/metabolismo , ARN Mensajero/metabolismo , Regulación hacia Arriba
18.
Mol Metab ; 54: 101334, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34487921

RESUMEN

OBJECTIVE: Protein disulfide isomerases (PDIs) are oxidoreductases that are involved in catalyzing the formation and rearrangement of disulfide bonds during protein folding. One of the PDI members is the PDI-associated 6 (PDIA6) protein, which has been shown to play a vital role in ß-cell dysfunction and diabetes. However, very little is known about the function of this protein in ß-cells in vivo. This study aimed to describe the consequences of a point mutation in Pdia6 on ß-cell development and function. METHODS: We generated an ENU mouse model carrying a missense mutation (Phe175Ser) in the second thioredoxin domain of the Pdia6 gene. Using biochemical and molecular tools, we determined the effects of the mutation on the ß-cell development at embryonic day (E)18.5 and ß-cell identity as well as function at postnatal stages. RESULTS: Mice homozygous for the Phe175Ser (F175S) mutation were mildly hyperglycemic at weaning and subsequently became hypoinsulinemic and overtly diabetic at the adult stage. Although no developmental phenotype was detected during embryogenesis, mutant mice displayed reduced insulin-expressing ß-cells at P14 and P21 without any changes in the rate of cell death and proliferation. Further analysis revealed an increase in BiP and the PDI family member PDIA4, but without any concomitant apoptosis and cell death. Instead, the expression of prominent markers of ß-cell maturation and function, such as Ins2, Mafa, and Slc2a2, along with increased expression of α-cell markers, Mafb, and glucagon was observed in adult mice, suggesting loss of ß-cell identity. CONCLUSIONS: The results demonstrate that a global Pdia6 mutation renders mice hypoinsulinemic and hyperglycemic. This occurs due to the loss of pancreatic ß-cell function and identity, suggesting a critical role of PDIA6 specifically for ß-cells.


Asunto(s)
Diabetes Mellitus/genética , Células Secretoras de Insulina/metabolismo , Proteína Disulfuro Isomerasas/genética , Animales , Diabetes Mellitus/metabolismo , Ratones , Ratones Endogámicos C3H , Mutación Puntual , Proteína Disulfuro Isomerasas/metabolismo
19.
Nat Commun ; 12(1): 674, 2021 01 29.
Artículo en Inglés | MEDLINE | ID: mdl-33514698

RESUMEN

Transcriptionally mature and immature ß-cells co-exist within the adult islet. How such diversity contributes to insulin release remains poorly understood. Here we show that subtle differences in ß-cell maturity, defined using PDX1 and MAFA expression, contribute to islet operation. Functional mapping of rodent and human islets containing proportionally more PDX1HIGH and MAFAHIGH ß-cells reveals defects in metabolism, ionic fluxes and insulin secretion. At the transcriptomic level, the presence of increased numbers of PDX1HIGH and MAFAHIGH ß-cells leads to dysregulation of gene pathways involved in metabolic processes. Using a chemogenetic disruption strategy, differences in PDX1 and MAFA expression are shown to depend on islet Ca2+ signaling patterns. During metabolic stress, islet function can be restored by redressing the balance between PDX1 and MAFA levels across the ß-cell population. Thus, preserving heterogeneity in PDX1 and MAFA expression, and more widely in ß-cell maturity, might be important for the maintenance of islet function.


Asunto(s)
Secreción de Insulina/fisiología , Células Secretoras de Insulina/metabolismo , Animales , Calcio/metabolismo , Células Cultivadas , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Femenino , Técnicas de Sustitución del Gen , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Factores de Transcripción Maf de Gran Tamaño/genética , Factores de Transcripción Maf de Gran Tamaño/metabolismo , Masculino , Ratones , Ratones Transgénicos , Modelos Animales , Cultivo Primario de Células , Transactivadores/genética , Transactivadores/metabolismo
20.
Nat Metab ; 2(2): 192-209, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-32694693

RESUMEN

Dedifferentiation of insulin-secreting ß cells in the islets of Langerhans has been proposed to be a major mechanism of ß-cell dysfunction. Whether dedifferentiated ß cells can be targeted by pharmacological intervention for diabetes remission, and ways in which this could be accomplished, are unknown as yet. Here we report the use of streptozotocin-induced diabetes to study ß-cell dedifferentiation in mice. Single-cell RNA sequencing (scRNA-seq) of islets identified markers and pathways associated with ß-cell dedifferentiation and dysfunction. Single and combinatorial pharmacology further show that insulin treatment triggers insulin receptor pathway activation in ß cells and restores maturation and function for diabetes remission. Additional ß-cell selective delivery of oestrogen by Glucagon-like peptide-1 (GLP-1-oestrogen conjugate) decreases daily insulin requirements by 60%, triggers oestrogen-specific activation of the endoplasmic-reticulum-associated protein degradation system, and further increases ß-cell survival and regeneration. GLP-1-oestrogen also protects human ß cells against cytokine-induced dysfunction. This study not only describes mechanisms of ß-cell dedifferentiation and regeneration, but also reveals pharmacological entry points to target dedifferentiated ß cells for diabetes remission.


Asunto(s)
Diabetes Mellitus Experimental/tratamiento farmacológico , Hipoglucemiantes/uso terapéutico , Células Secretoras de Insulina/patología , Insulina/uso terapéutico , Animales , Diabetes Mellitus Experimental/patología , Estrógenos/uso terapéutico , Péptido 1 Similar al Glucagón/uso terapéutico , Homeostasis , Humanos , Ratones , Polifarmacología , Inducción de Remisión , Estreptozocina
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