RESUMO
Dysfunction of pancreatic δ cells contributes to the etiology of diabetes. Despite their important role, human δ cells are scarce, limiting physiological studies and drug discovery targeting δ cells. To date, no directed δ-cell differentiation method has been established. Here, we demonstrate that fibroblast growth factor (FGF) 7 promotes pancreatic endoderm/progenitor differentiation, whereas FGF2 biases cells towards the pancreatic δ-cell lineage via FGF receptor 1. We develop a differentiation method to generate δ cells from human stem cells by combining FGF2 with FGF7, which synergistically directs pancreatic lineage differentiation and modulates the expression of transcription factors and SST activators during endoderm/endocrine precursor induction. These δ cells display mature RNA profiles and fine secretory granules, secrete somatostatin in response to various stimuli, and suppress insulin secretion from in vitro co-cultured ß cells and mouse ß cells upon transplantation. The generation of human pancreatic δ cells from stem cells in vitro would provide an unprecedented cell source for drug discovery and cell transplantation studies in diabetes.
Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes , Humanos , Animais , Camundongos , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Somatostatina/metabolismo , Células Secretoras de Somatostatina/citologia , Endoderma/citologia , Endoderma/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Pâncreas/citologia , Pâncreas/metabolismo , Somatostatina/metabolismo , Linhagem da Célula , Insulina/metabolismo , Secreção de InsulinaRESUMO
Glucagon-like peptide-1 receptor (GLP1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR) are transmembrane receptors involved in insulin, glucagon and somatostatin secretion from the pancreatic islet. Therapeutic targeting of GLP1R and GIPR restores blood glucose levels in part by influencing beta cell, alpha cell and delta cell function. Despite the importance of the incretin-mimetics for diabetes therapy, our understanding of GLP1R and GIPR expression patterns and signaling within the islet remain incomplete. Here, we present the evidence for GLP1R and GIPR expression in the major islet cell types, before addressing signaling pathway(s) engaged, as well as their influence on cell survival and function. While GLP1R is largely a beta cell-specific marker within the islet, GIPR is expressed in alpha cells, beta cells, and (possibly) delta cells. GLP1R and GIPR engage Gs-coupled pathways in most settings, although the exact outcome on hormone release depends on paracrine communication and promiscuous signaling. Biased agonism away from beta-arrestin is an emerging concept for improving therapeutic efficacy, and is also relevant for GLP1R/GIPR dual agonism. Lastly, dual agonists exert multiple effects on islet function through GIPR > GLP1R imbalance, increased GLP1R surface expression and cAMP signaling, as well as beneficial alpha cell-beta cell-delta cell crosstalk.
Assuntos
Células Secretoras de Glucagon , Receptores dos Hormônios Gastrointestinais , Células Secretoras de Somatostatina/metabolismo , Células Secretoras de Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Receptores dos Hormônios Gastrointestinais/metabolismo , Polipeptídeo Inibidor Gástrico/genética , Polipeptídeo Inibidor Gástrico/metabolismo , Transdução de SinaisRESUMO
Type 1 diabetes is an autoimmune disease that emerges with the destruction of beta cells of pancreatic Langerhans islets. Three different therapeutical approaches have been developed so far; pancreas transplantation, islet transplantation, and cell-based therapies. Bioengineering cell sheets for tissue generating is one of the latest approaches that have been used to construct cell-sheets instead of single cells so that it mimics the in vivo environments more. In this study, extra-hepatic functional islet tissue was constructed by transferring the 3-D beta cells and GFP labelled MSCs MSC sheets to the subcutaneous site of rats with STZ-induced diabetes. rBM-MSCs and beta cells were cultured on the 6-well PIPAAm culture dishes. Obtained rBM-MSCs as two-cell sheets and beta cells cultured in droplets with matrigel has transplanted into the dorsal subcutaneous area of diabetic rats. Fasting blood glucose levels and body weights were evaluated for 30 days after transplantation. Immunocytochemistry analysis for the anti-apoptotic, anti-inflammatory, and angiogenetic effects of MSCs on the 30th day of subcutaneous cell transplantation. All recipient rats transplanted with beta-cells with MSCs returned toward normoglycemia by day 5 and remained at this level for 30 days. Immunocytochemical analyses supported that the MSCs and beta cells preserved their viability and function. MSCs secrete cytokines and growth factors TGF-ß and IL-6; MSCs of the important features of the anti-apoptotic and anti-inflammatory properties, thanks to apoptosis of beta cells preserve graft explained by inhibition. In transplantation of MSCs induced angiogenesis and neovascularization, PECAM-1 and GFP positive simultaneously determining endothelial cells was observed indicating. Subcutaneous 3D beta-cell transplantation would be possible with the MSC-sheets as a feeder layer of beta cells. The beta-cell line is glucose-sensitive and has a high insulin release potential, and can be used as an alternative to islets in in vivo transplant studies.
Assuntos
Diabetes Mellitus Experimental , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Ratos , Animais , Células Endoteliais/metabolismo , Células Secretoras de Somatostatina/metabolismo , Transplante das Ilhotas Pancreáticas/fisiologia , Bioengenharia , Insulina/metabolismoRESUMO
Single-nucleus assay for transposase-accessible chromatin using sequencing (snATAC-seq) creates new opportunities to dissect cell type-specific mechanisms of complex diseases. Since pancreatic islets are central to type 2 diabetes (T2D), we profiled 15,298 islet cells by using combinatorial barcoding snATAC-seq and identified 12 clusters, including multiple alpha, beta and delta cell states. We cataloged 228,873 accessible chromatin sites and identified transcription factors underlying lineage- and state-specific regulation. We observed state-specific enrichment of fasting glucose and T2D genome-wide association studies for beta cells and enrichment for other endocrine cell types. At T2D signals localized to islet-accessible chromatin, we prioritized variants with predicted regulatory function and co-accessibility with target genes. A causal T2D variant rs231361 at the KCNQ1 locus had predicted effects on a beta cell enhancer co-accessible with INS and genome editing in embryonic stem cell-derived beta cells affected INS levels. Together our findings demonstrate the power of single-cell epigenomics for interpreting complex disease genetics.
Assuntos
Cromatina/química , Diabetes Mellitus Tipo 2/genética , Células Secretoras de Glucagon/metabolismo , Células Secretoras de Insulina/metabolismo , Canal de Potássio KCNQ1/genética , Células Secretoras de Polipeptídeo Pancreático/metabolismo , Células Secretoras de Somatostatina/metabolismo , Glicemia/metabolismo , Diferenciação Celular , Cromatina/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Epigenômica , Jejum , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Células Secretoras de Glucagon/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Células-Tronco Embrionárias Humanas/citologia , Humanos , Células Secretoras de Insulina/patologia , Canal de Potássio KCNQ1/metabolismo , Família Multigênica , Células Secretoras de Polipeptídeo Pancreático/patologia , Polimorfismo Genético , Análise de Célula Única , Células Secretoras de Somatostatina/patologia , Fatores de Transcrição/classificação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Somatostatin (SST) and somatostatin receptors (SSTRs) play an important role in the brain and gastrointestinal (GI) system. SST is produced in various organs and cells, and the inhibitory function of somatostatin-containing cells is involved in a range of physiological functions and pathological modifications. The GI system is the largest endocrine organ for digestion and absorption, SST-endocrine cells and neurons in the GI system are a critical effecter to maintain homeostasis via SSTRs 1-5 and co-receptors, while SST-SSTRs are involved in chemo-sensory, mucus, and hormone secretion, motility, inflammation response, itch, and pain via the autocrine, paracrine, endocrine, and exoendocrine pathways. It is also a power inhibitor for tumor cell proliferation, severe inflammation, and post-operation complications, and is a first-line anti-cancer drug in clinical practice. This mini review focuses on the current function of producing SST endocrine cells and local neurons SST-SSTRs in the GI system, discusses new development prognostic markers, phosphate-specific antibodies, and molecular imaging emerging in diagnostics and therapy, and summarizes the mechanism of the SST family in basic research and clinical practice. Understanding of endocrines and neuroendocrines in SST-SSTRs in GI will provide an insight into advanced medicine in basic and clinical research.
Assuntos
Trato Gastrointestinal/fisiologia , Receptores de Somatostatina/fisiologia , Somatostatina/fisiologia , Animais , Antineoplásicos/farmacologia , Comunicação Celular/efeitos dos fármacos , Proliferação de Células , Modelos Animais de Doenças , Sistema Nervoso Entérico/fisiologia , Homeostase , Humanos , Inflamação , Ligantes , Neurônios/metabolismo , Sistema Nervoso Parassimpático/fisiologia , Prognóstico , Receptores de Somatostatina/metabolismo , Somatostatina/metabolismo , Células Secretoras de Somatostatina/metabolismo , Sistema Nervoso Simpático/fisiologiaRESUMO
For much of the last century, our knowledge regarding the pancreas in type 1 and type 2 diabetes was largely derived from autopsy studies of individuals with these disorders or investigations utilising rodent models of either disease. While many important insights emanated from these efforts, the mode for investigation has increasingly seen change due to the availability of transplant-quality organ-donor tissues, improvements in pancreatic imaging, advances in metabolic assessments of living patients, genetic analyses, technological advances for laboratory investigation and more. As a result, many long-standing notions regarding the role for and the changes that occur in the pancreas in individuals with these disorders have come under question, while, at the same time, new issues (e.g., beta cell persistence, disease heterogeneity, exocrine contributions) have arisen. In this article, we will consider the vital role of the pancreas in human health and physiology, including discussion of its anatomical features and dual (exocrine and endocrine) functions. Specifically, we convey changes that occur in the pancreas of those with either type 1 or type 2 diabetes, with careful attention to the facets that may contribute to the pathogenesis of either disorder. Finally, we discuss the emerging unknowns with the belief that understanding the role of the pancreas in type 1 and type 2 diabetes will lead to improvements in disease diagnosis, understanding of disease heterogeneity and optimisation of treatments at a personalised level. Graphical abstract.
Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Pâncreas/metabolismo , Tecido Adiposo/patologia , Amiloidose/metabolismo , Amiloidose/patologia , Autoimunidade/imunologia , Diabetes Mellitus Tipo 1/imunologia , Células Secretoras de Glucagon/metabolismo , Células Secretoras de Glucagon/patologia , Humanos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/irrigação sanguínea , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Ilhotas Pancreáticas/fisiopatologia , Pâncreas/patologia , Pâncreas/fisiopatologia , Pâncreas Exócrino/metabolismo , Pâncreas Exócrino/patologia , Pâncreas Exócrino/fisiopatologia , Células Secretoras de Somatostatina/metabolismo , Células Secretoras de Somatostatina/patologiaRESUMO
OBJECTIVES: The main endocrine cell types in pancreatic islets are alpha, beta, and delta cells. Although these cell types have distinct roles in the regulation of glucose homeostasis, inadequate purification methods preclude the study of cell type-specific effects. We developed a reliable approach that enables simultaneous sorting of live alpha, beta, and delta cells from mouse islets for downstream analyses. METHODS: We developed an antibody panel against cell surface antigens to enable isolation of highly purified endocrine subsets from mouse islets based on the specific differential expression of CD71 on beta cells and CD24 on delta cells. We rigorously demonstrated the reliability and validity of our approach using bulk and single cell qPCR, immunocytochemistry, reporter mice, and transcriptomics. RESULTS: Pancreatic alpha, beta, and delta cells can be separated based on beta cell-specific CD71 surface expression and high expression of CD24 on delta cells. We applied our new sorting strategy to demonstrate that CD71, which is the transferrin receptor mediating the uptake of transferrin-bound iron, is upregulated in beta cells during early postnatal weeks. We found that beta cells express higher levels of several other genes implicated in iron metabolism and iron deprivation significantly impaired beta cell function. In human beta cells, CD71 is similarly required for iron uptake and CD71 surface expression is regulated in a glucose-dependent manner. CONCLUSIONS: This study provides a novel and efficient purification method for murine alpha, beta, and delta cells, identifies for the first time CD71 as a postnatal beta cell-specific marker, and demonstrates a central role of iron metabolism in beta cell function.
Assuntos
Antígenos de Superfície/imunologia , Células Secretoras de Insulina/metabolismo , Ferro/metabolismo , Animais , Antígenos CD/imunologia , Antígenos de Superfície/isolamento & purificação , Antígenos de Superfície/metabolismo , Biomarcadores/metabolismo , Antígeno CD24/imunologia , Linhagem Celular , Feminino , Células Secretoras de Glucagon/imunologia , Células Secretoras de Glucagon/metabolismo , Células Secretoras de Glucagon/fisiologia , Humanos , Imuno-Histoquímica/métodos , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/fisiologia , Ferro/fisiologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pâncreas/metabolismo , Pâncreas/fisiologia , Receptores da Transferrina/imunologia , Reprodutibilidade dos Testes , Células Secretoras de Somatostatina/imunologia , Células Secretoras de Somatostatina/metabolismo , Células Secretoras de Somatostatina/fisiologiaRESUMO
The pancreatic islet is a dense cellular network comprised of several cell types with endocrine function vital in the control of glucose homeostasis, metabolism, and feeding behavior. Within the islet, endocrine hormones also form an intricate paracrine network with supportive cells (endothelial, neuronal, immune) and secondary signaling molecules regulating cellular function and survival. Modulation of these signals has potential consequences for diabetes development, progression, and therapeutic intervention. Beta cell loss, reduced endogenous insulin secretion, and dysregulated glucagon secretion are hallmark features of both type 1 and 2 diabetes that not only impact systemic regulation of glucose, but also contribute to the function and survival of cells within the islet. Advancing research and technology have revealed new islet biology (cellular identity and transcriptomes) and identified previously unrecognized paracrine signals and mechanisms (somatostatin and ghrelin paracrine actions), while shifting prior views of intraislet communication. This review will summarize the paracrine signals regulating islet endocrine function and survival, the disruption and dysfunction that occur in diabetes, and potential therapeutic targets to preserve beta cell mass and function.
Assuntos
Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Comunicação Parácrina , Transdução de Sinais , Animais , Sobrevivência Celular , Grelina/metabolismo , Células Secretoras de Glucagon/metabolismo , Glucose/metabolismo , Glicogênio/metabolismo , Humanos , Ilhotas Pancreáticas/citologia , Polipeptídeo Pancreático/metabolismo , Somatostatina/metabolismo , Células Secretoras de Somatostatina/metabolismoRESUMO
Insulin-producing cells derived from in vitro differentiation of stem cells and non-stem cells by using different factors can spare the need for genetic manipulation and provide a cure for diabetes. In this context, pancreatic progenitors differentiating to ß-like cells garner increasing attention as ß-cell replacement source. This kind of cell therapy has the potential to cure diabetes, but is still on its way of being clinically useful. The primary restriction for in vitro production of mature and functional ß-cells is developing a physiologically relevant in vitro culture system which can mimic in vivo pathways of islet development. In order to achieve this target, different approaches have been attempted for the differentiation of pancreatic stem/progenitor cells to ß-like cells. Here, we will review some of the state-of-the-art protocols for the differentiation of pancreatic progenitors and differentiated pancreatic cells into ß-like cells with a focus on pancreatic duct cells.
Assuntos
Células Acinares/metabolismo , Diferenciação Celular , Células Secretoras de Glucagon/metabolismo , Células Secretoras de Insulina/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células Secretoras de Somatostatina/metabolismo , Células Acinares/transplante , Animais , Diabetes Mellitus/terapia , Células Secretoras de Glucagon/transplante , Humanos , Insulina/metabolismo , Transplante das Ilhotas Pancreáticas , Transplante de Células-Tronco MesenquimaisRESUMO
Little is known about the role of islet delta cells in regulating blood glucose homeostasis in vivo. Delta cells are important paracrine regulators of beta cell and alpha cell secretory activity, however the structural basis underlying this regulation has yet to be determined. Most delta cells are elongated and have a well-defined cell soma and a filopodia-like structure. Using in vivo optogenetics and high-speed Ca2+ imaging, we show that these filopodia are dynamic structures that contain a secretory machinery, enabling the delta cell to reach a large number of beta cells within the islet. This provides for efficient regulation of beta cell activity and is modulated by endogenous IGF-1/VEGF-A signaling. In pre-diabetes, delta cells undergo morphological changes that may be a compensation to maintain paracrine regulation of the beta cell. Our data provides an integrated picture of how delta cells can modulate beta cell activity under physiological conditions.
Assuntos
Ilhotas Pancreáticas/ultraestrutura , Comunicação Parácrina , Estado Pré-Diabético/patologia , Pseudópodes/ultraestrutura , Células Secretoras de Somatostatina/ultraestrutura , Animais , Glicemia/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/ultraestrutura , Microscopia Intravital , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Imagem Óptica , Optogenética , Estado Pré-Diabético/metabolismo , Pseudópodes/metabolismo , Células Secretoras de Somatostatina/citologia , Células Secretoras de Somatostatina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Somatostatin secretion from pancreatic islet δ-cells is stimulated by elevated glucose levels, but the underlying mechanisms have only partially been elucidated. Here we show that glucose-induced somatostatin secretion (GISS) involves both membrane potential-dependent and -independent pathways. Although glucose-induced electrical activity triggers somatostatin release, the sugar also stimulates GISS via a cAMP-dependent stimulation of CICR and exocytosis of somatostatin. The latter effect is more quantitatively important and in mouse islets depolarized by 70 mM extracellular K+ , increasing glucose from 1 mM to 20 mM produced an â¼3.5-fold stimulation of somatostatin secretion, an effect that was mimicked by the application of the adenylyl cyclase activator forskolin. Inhibiting cAMP-dependent pathways with PKI or ESI-05, which inhibit PKA and exchange protein directly activated by cAMP 2 (Epac2), respectively, reduced glucose/forskolin-induced somatostatin secretion. Ryanodine produced a similar effect that was not additive to that of the PKA or Epac2 inhibitors. Intracellular application of cAMP produced a concentration-dependent stimulation of somatostatin exocytosis and elevation of cytoplasmic Ca2+ ([Ca2+]i). Both effects were inhibited by ESI-05 and thapsigargin (an inhibitor of SERCA). By contrast, inhibition of PKA suppressed δ-cell exocytosis without affecting [Ca2+]i Simultaneous recordings of electrical activity and [Ca2+]i in δ-cells expressing the genetically encoded Ca2+ indicator GCaMP3 revealed that the majority of glucose-induced [Ca2+]i spikes did not correlate with δ-cell electrical activity but instead reflected Ca2+ release from the ER. These spontaneous [Ca2+]i spikes are resistant to PKI but sensitive to ESI-05 or thapsigargin. We propose that cAMP links an increase in plasma glucose to stimulation of somatostatin secretion by promoting CICR, thus evoking exocytosis of somatostatin-containing secretory vesicles in the δ-cell.
Assuntos
Cálcio/metabolismo , AMP Cíclico/metabolismo , Glucose/farmacologia , Pâncreas/citologia , Células Secretoras de Somatostatina/efeitos dos fármacos , Somatostatina/metabolismo , Adjuvantes Imunológicos/farmacologia , Animais , Membrana Celular/fisiologia , Colforsina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Células Secretoras de Somatostatina/metabolismo , Tapsigargina/farmacologiaRESUMO
Although kisspeptin is the primary stimulator of gonadotropin-releasing hormone secretion and therefore the hypothalamic-pituitary-gonadal axis, recent findings suggest kisspeptin can also regulate additional neuroendocrine processes including release of growth hormone (GH). Here we show that central delivery of kisspeptin causes a robust rise in plasma GH in fasted but not fed sheep. Kisspeptin-induced GH secretion was similar in animals fasted for 24 hours and those fasted for 72 hours, suggesting that the factors involved in kisspeptin-induced GH secretion are responsive to loss of food availability and not the result of severe negative energy balance. Pretreatment with the neuropeptide Y (NPY) Y1 receptor antagonist, BIBO 3304, blocked the effects of kisspeptin-induced GH release, implicating NPY as an intermediary. Kisspeptin treatment induced c-Fos in NPY and GH-releasing hormone (GHRH) cells of the arcuate nucleus. The same kisspeptin treatment resulted in a reduction in c-Fos in somatostatin (SS) cells in the periventricular nucleus. Finally, blockade of systemic ghrelin release or antagonism of the ghrelin receptor eliminated or reduced the ability of kisspeptin to induce GH release, suggesting the presence of ghrelin is required for kisspeptin-induced GH release in fasted animals. Our findings support the hypothesis that during short-term fasting, systemic ghrelin concentrations and NPY expression in the arcuate nucleus rise. This permits kisspeptin activation of NPY cells. In turn, NPY stimulates GHRH cells and inhibits SS cells, resulting in GH release. We propose a mechanism by which kisspeptin conveys reproductive and hormone status onto the somatotropic axis, resulting in alterations in GH release.
Assuntos
Grelina/metabolismo , Hormônio do Crescimento/efeitos dos fármacos , Kisspeptinas/farmacologia , Neuropeptídeo Y/metabolismo , Células Secretoras de Somatostatina/efeitos dos fármacos , Animais , Núcleo Arqueado do Hipotálamo/efeitos dos fármacos , Núcleo Arqueado do Hipotálamo/metabolismo , Arginina/análogos & derivados , Arginina/farmacologia , Atropina/farmacologia , Jejum/metabolismo , Feminino , Imunofluorescência , Hormônio do Crescimento/metabolismo , Hormônio Liberador de Hormônio do Crescimento , Antagonistas Muscarínicos/farmacologia , Oligopeptídeos/farmacologia , Proteínas Proto-Oncogênicas c-fos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Receptores de Grelina/antagonistas & inibidores , Receptores de Neuropeptídeo Y/antagonistas & inibidores , Ovinos , Carneiro Doméstico , Células Secretoras de Somatostatina/metabolismoRESUMO
The progressive loss of pancreatic ß-cell mass that occurs in both type 1 and type 2 diabetes is a primary factor driving efforts to identify strategies for effectively increasing, enhancing or restoring ß-cell mass. While factors that seem to influence ß-cell proliferation in specific contexts have been described, reliable stimulation of human ß-cell proliferation has remained a challenge. Importantly, ß-cells exist in the context of a complex, integrated pancreatic islet microenvironment where they interact with other endocrine cells, vascular endothelial cells, extracellular matrix, neuronal projections and islet macrophages. This review highlights different components of the pancreatic microenvironment, and reviews what is known about how signaling that occurs between ß-cells and these other components influences ß-cell proliferation. Future efforts to further define the role of the pancreatic islet microenvironment on ß-cell proliferation may lead to the development of successful approaches to increase or restore ß-cell mass in diabetes.
Assuntos
Comunicação Celular , Proliferação de Células , Microambiente Celular , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Modelos Biológicos , Animais , Apoptose , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Matriz Extracelular/imunologia , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Células Secretoras de Glucagon/citologia , Células Secretoras de Glucagon/imunologia , Células Secretoras de Glucagon/metabolismo , Células Secretoras de Glucagon/patologia , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/patologia , Ilhotas Pancreáticas/irrigação sanguínea , Ilhotas Pancreáticas/inervação , Ilhotas Pancreáticas/patologia , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Células Secretoras de Polipeptídeo Pancreático/citologia , Células Secretoras de Polipeptídeo Pancreático/imunologia , Células Secretoras de Polipeptídeo Pancreático/metabolismo , Células Secretoras de Polipeptídeo Pancreático/patologia , Células Secretoras de Somatostatina/citologia , Células Secretoras de Somatostatina/imunologia , Células Secretoras de Somatostatina/metabolismo , Células Secretoras de Somatostatina/patologia , Especificidade da EspécieRESUMO
Somatostatin secreted by pancreatic δ cells mediates important paracrine interactions in Langerhans islets, including maintenance of glucose metabolism through the control of reciprocal insulin and glucagon secretion. Disruption of this circuit contributes to the development of diabetes. However, the precise mechanisms that control somatostatin secretion from islets remain elusive. Here, we found that a super-complex comprising the cullin 4B-RING E3 ligase (CRL4B) and polycomb repressive complex 2 (PRC2) epigenetically regulates somatostatin secretion in islets. Constitutive ablation of CUL4B, the core component of the CRL4B-PRC2 complex, in δ cells impaired glucose tolerance and decreased insulin secretion through enhanced somatostatin release. Moreover, mechanistic studies showed that the CRL4B-PRC2 complex, under the control of the δ cell-specific transcription factor hematopoietically expressed homeobox (HHEX), determines the levels of intracellular calcium and cAMP through histone posttranslational modifications, thereby altering expression of the Cav1.2 calcium channel and adenylyl cyclase 6 (AC6) and modulating somatostatin secretion. In response to high glucose levels or urocortin 3 (UCN3) stimulation, increased expression of cullin 4B (CUL4B) and the PRC2 subunit histone-lysine N-methyltransferase EZH2 and reciprocal decreases in Cav1.2 and AC6 expression were found to regulate somatostatin secretion. Our results reveal an epigenetic regulatory mechanism of δ cell paracrine interactions in which CRL4B-PRC2 complexes, Cav1.2, and AC6 expression fine-tune somatostatin secretion and facilitate glucose homeostasis in pancreatic islets.
Assuntos
Proteínas Culina/metabolismo , Insulina/metabolismo , Complexos Multienzimáticos/metabolismo , Comunicação Parácrina , Células Secretoras de Somatostatina/metabolismo , Somatostatina/metabolismo , Adenilil Ciclases/genética , Adenilil Ciclases/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Proteínas Culina/genética , AMP Cíclico/metabolismo , Epigênese Genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Insulina/genética , Secreção de Insulina , Camundongos , Camundongos Knockout , Complexos Multienzimáticos/genética , Somatostatina/genética , Células Secretoras de Somatostatina/citologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Glucagon is the main counterregulatory hormone in the body. Still, the mechanism involved in the regulation of glucagon secretion from pancreatic alpha cells remains elusive. Dysregulated glucagon secretion is common in patients with Cystic Fibrosis (CF) that develop CF related diabetes (CFRD). CF is caused by a mutation in the Cl- channel Cystic fibrosis transmembrane conductance regulator (CFTR), but whether CFTR is present in human alpha cells and regulate glucagon secretion has not been investigated in detail. Here, both human and mouse alpha cells showed CFTR protein expression, whereas CFTR was absent in somatostatin secreting delta cells. CFTR-current activity induced by cAMP was measured in single alpha cells. Glucagon secretion at different glucose levels and in the presence of forskolin was increased by CFTR-inhibition in human islets, whereas depolarization-induced glucagon secretion was unaffected. CFTR is suggested to mainly regulate the membrane potential through an intrinsic alpha cell effect, as supported by a mathematical model of alpha cell electrophysiology. In conclusion, CFTR channels are present in alpha cells and act as important negative regulators of cAMP-enhanced glucagon secretion through effects on alpha cell membrane potential. Our data support that loss-of-function mutations in CFTR contributes to dysregulated glucagon secretion in CFRD.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Secretoras de Glucagon/citologia , Glucagon/metabolismo , Animais , Células Cultivadas , Colforsina/metabolismo , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Feminino , Células Secretoras de Glucagon/efeitos dos fármacos , Células Secretoras de Glucagon/metabolismo , Glucose/farmacologia , Humanos , Masculino , Potenciais da Membrana , Camundongos , Pessoa de Meia-Idade , Mutação , Células Secretoras de Somatostatina/citologia , Células Secretoras de Somatostatina/efeitos dos fármacos , Células Secretoras de Somatostatina/metabolismoRESUMO
SerpinI2/Pancpin/MEPI is a 46kDa member of the serpin (serine protease inhibitor) superfamily. It is downregulated in pancreatic and breast cancer, and associated with acinar cell apoptosis and pancreatic insufficiency when absent in mice. However, the target protease and protein properties of serpinI2 are previously uncharacterised. We have expressed and purified recombinant serpin I2 in E. coli. The protein exhibited thermal instability typical of inhibitory serpins, which was lost following RCL cleavage. SerpinI2 did not inhibit trypsin, but was found to inhibit pancreatic chymotrypsin and elastase with Kass values >105M-1s-1, and with stoichiometry of inhibition of 1.4 and 1.7 respectively. Mutagenesis of the predicted critical hinge region residue Ser344 abolished inhibitory activity, and a cleavage site C-terminal to Met358 was identified. The protein is also prone to polymerisation/aggregation at 45°C, a characteristic of serpins associated with disease. This study therefore reveals a function for serpinI2 and supports the hypothesis that this protein can protect pancreatic cells from prematurely activated zymogens.
Assuntos
Quimotripsina/antagonistas & inibidores , Elastase Pancreática/antagonistas & inibidores , Inibidores de Serina Proteinase/farmacologia , Serpinas/farmacologia , Sequência de Aminoácidos , Linhagem Celular , Escherichia coli/metabolismo , Proteínas de Neoplasias/farmacologia , Proteínas Recombinantes/farmacologia , Homologia de Sequência de Aminoácidos , Células Secretoras de Somatostatina/efeitos dos fármacos , Células Secretoras de Somatostatina/metabolismo , Especificidade por Substrato , Tripsina/metabolismoRESUMO
During embryonic development, endocrine cells of the pancreas are specified from multipotent progenitors. The transcription factor Neurogenin 3 (NEUROG3) is critical for this development and it has been shown that all endocrine cells of the pancreas arise from endocrine progenitors expressing NEUROG3. A thorough understanding of the role of NEUROG3 during development, directed differentiation of pluripotent stem cells and in models of cellular reprogramming, will guide future efforts directed at finding novel sources of ß-cells for cell replacement therapies. In this article, we review the expression and function of NEUROG3 in both mouse and human and present the further characterization of a monoclonal antibody directed against NEUROG3. This antibody has been previously been used for detection of both mouse and human NEUROG3. However, our results suggest that the epitope recognized by this antibody is specific to mouse NEUROG3. Thus, we have also generated a monoclonal antibody specifically recognizing human NEUROG3 and present the characterization of this antibody here. Together, these antibodies will provide useful tools for future studies of NEUROG3 expression, and the data presented in this article suggest that recently described expression patterns of NEUROG3 in human foetal and adult pancreas should be re-examined.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Ilhotas Pancreáticas/citologia , Proteínas do Tecido Nervoso/genética , Animais , Anticorpos Monoclonais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Reprogramação Celular , Células Secretoras de Glucagon/citologia , Células Secretoras de Glucagon/metabolismo , Humanos , Imuno-Histoquímica , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Células Secretoras de Polipeptídeo Pancreático/citologia , Células Secretoras de Polipeptídeo Pancreático/metabolismo , Células Secretoras de Somatostatina/citologia , Células Secretoras de Somatostatina/metabolismoRESUMO
The aim of this study was to evaluate the location of PP and δ cells in relation to the vascularization within human pancreatic islets. To this end, pancreas sections were analysed by immunofluorescence using antibodies against endocrine islet and endothelial cells. Staining in different islet areas corresponding to islet cells adjacent or not to peripheral or central vascular channels was quantified by computerized morphometry. As results, α, PP and δ cells were preferentially found adjacent to vessels. In contrast to α cells, which were evenly distributed between islet periphery and intraislet vascular channels, PP and δ cells had asymmetric and opposite distributions: PP staining was higher and somatostatin staining was lower in the islet periphery than in the area around intraislet vascular channels. Additionally, frequencies of PP and δ cells were negatively correlated in the islets. No difference was observed between islets from the head and the tail of the pancreas, and from type 2 diabetic and non-diabetic donors. In conclusion, the distribution of δ cells differs from that of PP cells in human islets, suggesting that vessels at the periphery and at the centre of islets drain different hormonal cocktails.
Assuntos
Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Células Secretoras de Polipeptídeo Pancreático/citologia , Células Secretoras de Polipeptídeo Pancreático/metabolismo , Células Secretoras de Somatostatina/citologia , Células Secretoras de Somatostatina/metabolismo , Adolescente , Adulto , Idoso , Imunofluorescência , Humanos , Pessoa de Meia-Idade , Polipeptídeo Pancreático/metabolismo , Somatostatina/metabolismo , Distribuição Tecidual , Adulto JovemRESUMO
The transcription factor Pax6 is an important regulator of development and cell differentiation in various organs. Thus, Pax6 was shown to promote neural development in the cerebral cortex and spinal cord, and to control pancreatic endocrine cell genesis. However, the role of Pax6 in distinct endocrine cells of the adult pancreas has not been addressed. We report the conditional inactivation of Pax6 in insulin and glucagon producing cells of the adult mouse pancreas. In the absence of Pax6, beta- and alpha-cells lose their molecular maturation characteristics. Our findings provide strong evidence that Pax6 is responsible for the maturation of beta-, and alpha-cells, but not of delta-, and PP-cells. Moreover, lineage-tracing experiments demonstrate that Pax6-deficient beta- and alpha-cells are shunted towards ghrelin marked cells, sustaining the idea that ghrelin may represent a marker for endocrine cell maturation.