RESUMO
Parathyroid hormone-related protein (PTHrP) is a pleiotropic hormone essential for morphogenesis, tissue differentiation, as well as cell regulation and function. PTHrP is expressed by pancreatic beta cells which are responsible for insulin secretion. Previous studies have reported that N-terminal PTHrP stimulated proliferation in beta cells in rodents. We have developed a knockin mouse model (PTHrP Δ/Δ) lacking the C-terminal and nuclear localization sequence (NLS) of PTHrP. These mice die at â¼day 5, are severely stunted in growth, weigh 54% less than control mice at day 1-2 and eventually fail to grow. PTHrP Δ/Δ mice are also hypoinsulinemic and hypoglycemic yet have nutrient intake proportional to size. To characterize the pancreatic islets in these mice, islets (â¼10-20) were isolated from 2 to 5 day-old-mice using collagenase digestion. Islets from PTHrP Δ/Δ mice were smaller in size but secreted more insulin than littermate controls. PTHrP Δ/Δ and control mice islets were exposed to various glucose concentrations and intracellular calcium, the trigger for insulin release, was elevated for glucose concentrations of 8-20 mM. Immunofluorescence staining showed less glucagon-stained area in islets from PTHrP Δ/Δ mice (â¼250 µm2) compared to islets from control mice (â¼900 µm2), and ELISA confirmed there was reduced glucagon content. These data collectively demonstrate increased insulin secretion and reduced glucagon at the islet level, which may contribute to the observed hypoglycemia and early death in PTHrP Δ/Δ mice. Thus, the C-terminus and NLS of PTHrP are crucial to life, including regulation of glucose homeostasis and islet function.
Assuntos
Ilhotas Pancreáticas , Proteína Relacionada ao Hormônio Paratireóideo , Animais , Camundongos , Glucagon , Glucose/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/crescimento & desenvolvimento , Ilhotas Pancreáticas/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo/genética , Proteína Relacionada ao Hormônio Paratireóideo/metabolismoRESUMO
Recently, we reported that forkhead box A2 (FOXA2) is required for the development of human pancreatic α- and ß-cells. However, whether miRNAs play a role in regulating pancreatic genes during pancreatic development in the absence of FOXA2 expression is largely unknown. Here, we aimed to capture the dysregulated miRNAs and to identify their pancreatic-specific gene targets in pancreatic progenitors (PPs) derived from wild-type induced pluripotent stem cells (WT-iPSCs) and from iPSCs lacking FOXA2 (FOXA2-/-iPSCs). To identify differentially expressed miRNAs (DEmiRs), and genes (DEGs), two different FOXA2-/-iPSC lines were differentiated into PPs. FOXA2-/- PPs showed a significant reduction in the expression of the main PP transcription factors (TFs) in comparison to WT-PPs. RNA sequencing analysis demonstrated significant reduction in the mRNA expression of genes involved in the development and function of exocrine and endocrine pancreas. Furthermore, miRNA profiling identified 107 downregulated and 111 upregulated DEmiRs in FOXA2-/- PPs compared to WT-PPs. Target prediction analysis between DEmiRs and DEGs identified 92 upregulated miRNAs, predicted to target 1498 downregulated genes in FOXA2-/- PPs. Several important pancreatic TFs essential for pancreatic development were targeted by multiple DEmiRs. Selected DEmiRs and DEGs were further validated using RT-qPCR. Our findings revealed that FOXA2 expression is crucial for pancreatic development through regulating the expression of pancreatic endocrine and exocrine genes targeted by a set of miRNAs at the pancreatic progenitor stage. These data provide novel insights of the effect of FOXA2 deficiency on miRNA-mRNA regulatory networks controlling pancreatic development and differentiation.
Assuntos
Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Fator 3-beta Nuclear de Hepatócito , Células-Tronco Pluripotentes Induzidas , Ilhotas Pancreáticas , MicroRNAs , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 3-beta Nuclear de Hepatócito/genética , Fator 3-beta Nuclear de Hepatócito/fisiologia , MicroRNAs/genética , Humanos , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/crescimento & desenvolvimento , Ilhotas Pancreáticas/metabolismo , Diferenciação Celular/genética , Linhagem CelularRESUMO
Delineating gene regulatory networks that orchestrate cell-type specification is a continuing challenge for developmental biologists. Single-cell analyses offer opportunities to address these challenges and accelerate discovery of rare cell lineage relationships and mechanisms underlying hierarchical lineage decisions. Here, we describe the molecular analysis of mouse pancreatic endocrine cell differentiation using single-cell transcriptomics, chromatin accessibility assays coupled to genetic labeling, and cytometry-based cell purification. We uncover transcription factor networks that delineate ß-, α-, and δ-cell lineages. Through genomic footprint analysis, we identify transcription factor-regulatory DNA interactions governing pancreatic cell development at unprecedented resolution. Our analysis suggests that the transcription factor Neurog3 may act as a pioneer transcription factor to specify the pancreatic endocrine lineage. These findings could improve protocols to generate replacement endocrine cells from renewable sources, like stem cells, for diabetes therapy.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Cromatina , Ilhotas Pancreáticas , Proteínas do Tecido Nervoso , Transcriptoma , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Cromatina/genética , Cromatina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Ilhotas Pancreáticas/crescimento & desenvolvimento , Ilhotas Pancreáticas/metabolismo , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Análise de Célula ÚnicaRESUMO
Glucose homeostasis is maintained by hormones secreted from different types of pancreatic islets and its dysregulation can result in diseases including diabetes mellitus. The secretion of hormones from pancreatic islets is highly complex and tightly controlled by G protein-coupled receptors (GPCRs). Moreover, GPCR signaling may play a role in enhancing islet cell replication and proliferation. Thus, targeting GPCRs offers a promising strategy for regulating the functionality of pancreatic islets. Here, available RNAseq datasets from human and mouse islets were used to identify the GPCR expression profile and the impact of GPCR signaling for normal islet functionality is discussed.
Assuntos
Ilhotas Pancreáticas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Células Secretoras de Glucagon/citologia , Células Secretoras de Glucagon/metabolismo , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/crescimento & desenvolvimento , Células Secretoras de Polipeptídeo Pancreático/citologia , Células Secretoras de Polipeptídeo Pancreático/metabolismo , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais , TranscriptomaRESUMO
Islet function is critical for normal glucose homeostasis. Unlike adult ß cells, fetal and neonatal islets are more proliferative and have decreased insulin secretion in response to stimuli. However, the underlying mechanisms governing functional maturity of islets have not been completely elucidated. Pancreatic islets comprise different cell types. The microenvironment of islets and interactions between these cell types are critical for ß-cell development and maturation. Thus, the study of intact islets is optimal to identify novel molecular mechanisms controlling islet functional development. Transcriptomes and genome-wide histone landscapes of H3K4me3, H3K27me3, and H3K27Ac from intact islets isolated from 2- and 10-week-old Sprague-Dawley rats were integrated to elucidate genes and pathways modulating islet development, as well as the contribution of epigenetic regulation. A total of 4489 differentially expressed genes were identified; 2289 and 2200 of them were up- and down-regulated in 10-week islets, respectively. Ingenuity Pathway Analysis revealed critical pathways regulating functional maturation of islets, including nutrient sensing, neuronal function, immune function, cell replication, and extracellular matrix. Furthermore, we identified significant changes in enrichment of H3K4me3, H3K27me3, and H3K27Ac marks, which correlated with expression changes of genes critical for islet function. These histone marks were enriched at critical transcription factor-binding motifs, such as Hoxa9, C/EBP-ß, Gata1, Foxo1, E2f1, E2f3, and Mafb. In addition, our chromatin immunoprecipitation sequencing data revealed multiple potential bivalent genes whose poised states changed with maturation. Collectively, our current study identified critical novel pathways for mature islet function and suggested a role for histone modifications in regulating islet development and maturation.
Assuntos
Diferenciação Celular/genética , Células Secretoras de Insulina/fisiologia , Ilhotas Pancreáticas/crescimento & desenvolvimento , Animais , Microambiente Celular/genética , Metabolismo Energético/genética , Epigênese Genética/fisiologia , Epigenoma/fisiologia , Regulação da Expressão Gênica , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/inervação , Ilhotas Pancreáticas/fisiologia , Ratos , Ratos Sprague-Dawley , Transcriptoma/fisiologiaRESUMO
BACKGROUND: Prenatal stress is associated with increased susceptibility to psychiatric and metabolic disorders later in life. Prenatal exposure to stress mediators may have sex-dependent effects on offspring brain and metabolic function, promoting a sex-specific vulnerability to psychopathology and metabolic alterations at adulthood. In this work, the impact of prenatal stress on glucose homeostasis and peripheral metabolism of male and female offspring was investigated in a chronic anxiety animal model. METHODS: Pregnant Wistar rats were injected with saline or glucocorticoid (dexamethasone: 1 mg/kg, subcutaneous) at gestational days 18 and 19. Male and female offspring weight was monitored, and anxious-like behaviour and peripheral insulin-sensitive tissues were analysed at adulthood. RESULTS: At birth, females and males prenatally exposed to stress presented decreased body weight which remained low in females. At adulthood, a morphological disorganization of the Langerhans islets was observed in both sexes prenatally exposed to stress, yet not changes in insulin levels were detected. Also, prenatal stress increased glucose transporter 4 (GLUT-4) levels in female and male adipose tissues and decreased insulin receptor levels in the liver and skeleton muscle but only in females. CONCLUSIONS: Exposure to stress mediators in critical periods of development negatively affects behaviour and metabolism. Prenatal stress programmes offspring peripheral metabolism in a sex-specific manner, emphasizing that the response to stress in critical periods of development may be sex-specific having each sex different vulnerabilities to psychiatric and metabolic disorders. Considering sex-specificities may provide critical clues for the design of preventive strategies and for early therapeutic intervention.
Assuntos
Ansiedade/metabolismo , Glucose/metabolismo , Complicações na Gravidez/metabolismo , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Estresse Psicológico/metabolismo , Tecido Adiposo/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Transportador de Glucose Tipo 4/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/crescimento & desenvolvimento , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Fígado/metabolismo , Masculino , Músculo Esquelético/metabolismo , Gravidez , Ratos , Receptor de Insulina/metabolismo , Fatores SexuaisRESUMO
In the endocrine pancreas, growth hormone (GH) is known to promote pancreatic islet growth and insulin secretion. In this study, we show that GH receptor (GHR) loss in the germline and in adulthood impacts islet mass in general but more profoundly in male mice. GHR knockout (GHRKO) mice have enhanced insulin sensitivity and low circulating insulin. We show that the total cross-sectional area of isolated islets (estimated islet mass) was reduced by 72% in male but by only 29% in female GHRKO mice compared with wild-type controls. Also, islets from GHRKO mice secreted â¼50% less glucose-stimulated insulin compared with size-matched islets from wild-type mice. We next used mice with a floxed Ghr gene to knock down the GHR in adult mice at 6 mo of age (6mGHRKO) and examined the impact on glucose and islet metabolism. By 12 mo of age, female 6mGHRKO mice had increased body fat and reduced islet mass but had no change in glucose tolerance or insulin sensitivity. However, male 6mGHRKO mice had nearly twice as much body fat, substantially reduced islet mass, and enhanced insulin sensitivity, but no change in glucose tolerance. Despite large losses in islet mass, glucose-stimulated insulin secretion from isolated islets was not significantly different between male 6mGHRKO and controls, whereas isolated islets from female 6mGHRKO mice showed increased glucose-stimulated insulin release. Our findings demonstrate the importance of GH to islet mass throughout life and that unique sex-specific adaptations to the loss of GH signaling allow mice to maintain normal glucose metabolism.NEW & NOTEWORTHY Growth hormone (GH) is important for more than just growth. GH helps to maintain pancreatic islet mass and insulin secretion throughout life. Sex-specific adaptations to the loss of GH signaling allow mice to maintain normal glucose regulation despite losing islet mass.
Assuntos
Células Germinativas/metabolismo , Hormônio do Crescimento/deficiência , Ilhotas Pancreáticas/crescimento & desenvolvimento , Ilhotas Pancreáticas/fisiologia , Receptores da Somatotropina/genética , Fatores Etários , Animais , Proliferação de Células/genética , Feminino , Células Germinativas/fisiologia , Hormônio do Crescimento/genética , Hormônio do Crescimento/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tamanho do Órgão/genética , Receptores da Somatotropina/deficiência , Receptores da Somatotropina/metabolismo , Caracteres Sexuais , Transdução de Sinais/genéticaRESUMO
Type 2 diabetes (T2D) has a strong genetic component. Most of the gene variants driving the pathogenesis of T2D seem to target pancreatic ß-cell function. To identify novel gene variants acting at early stage of the disease, we analyzed whole transcriptome data to identify differential expression (DE) and alternative exon splicing (AS) transcripts in pancreatic islets collected from two metabolically diverse mouse strains at 6 weeks of age after three weeks of high-fat-diet intervention. Our analysis revealed 1218 DE and 436 AS genes in islets from NZO/Hl vs C3HeB/FeJ. Whereas some of the revealed genes present well-established markers for ß-cell failure, such as Cd36 or Aldh1a3, we identified numerous DE/AS genes that have not been described in context with ß-cell function before. The gene Lgals2, previously associated with human T2D development, was DE as well as AS and localizes in a quantitative trait locus (QTL) for blood glucose on Chr.15 that we reported recently in our N2(NZOxC3H) population. In addition, pathway enrichment analysis of DE and AS genes showed an overlap of only half of the revealed pathways, indicating that DE and AS in large parts influence different pathways in T2D development. PPARG and adipogenesis pathways, two well-established metabolic pathways, were overrepresented for both DE and AS genes, probably as an adaptive mechanism to cope for increased cellular stress. Our results provide guidance for the identification of novel T2D candidate genes and demonstrate the presence of numerous AS transcripts possibly involved in islet function and maintenance of glucose homeostasis.
Assuntos
Diabetes Mellitus Tipo 2/genética , Galectina 2/genética , Insulina/genética , PPAR gama/genética , Adipogenia/genética , Processamento Alternativo/genética , Animais , Glicemia/genética , Antígenos CD36/genética , Diabetes Mellitus Tipo 2/patologia , Éxons/genética , Regulação da Expressão Gênica/genética , Humanos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Ilhotas Pancreáticas/crescimento & desenvolvimento , Ilhotas Pancreáticas/patologia , Redes e Vias Metabólicas/genética , Camundongos , Locos de Características Quantitativas/genética , Retinal Desidrogenase/genética , Transcriptoma/genéticaRESUMO
BACKGROUND: Growth-restricted fetuses have attenuated glucose-stimulated insulin secretion (GSIS), smaller pancreatic islets, less pancreatic ß-cells, and less pancreatic vascularization compared with normally growing fetuses. Infusion of leucine into normal late-gestation fetal sheep potentiates GSIS, as well as increases pancreatic islet size, the proportion of the pancreas and islet comprising ß-cells, and pancreatic and islet vascularity. In addition, leucine stimulates hepatocyte growth factor (HGF ) mRNA expression in islet endothelial cells isolated from normal fetal sheep. OBJECTIVE: We hypothesized that a 9-d leucine infusion would potentiate GSIS and increase pancreatic islet size, ß-cells, and vascularity in intrauterine fetal growth restriction (IUGR) fetal sheep. We also hypothesized that leucine would stimulate HGF mRNA in islet endothelial cells isolated from IUGR fetal sheep. METHODS: Late-gestation Columbia-Rambouillet IUGR fetal sheep (singleton or twin) underwent surgeries to place vascular sampling and infusion catheters. Fetuses were randomly allocated to receive a 9-d leucine infusion to achieve a 50-100% increase in leucine concentrations or a control saline infusion. GSIS was measured and pancreas tissue was processed for histologic analysis. Pancreatic islet endothelial cells were isolated from IUGR fetal sheep and incubated with supplemental leucine. Data were analyzed by mixed-models ANOVA; Student, Mann-Whitney, or a paired t test; or a test of equality of proportions. RESULTS: Chronic leucine infusion in IUGR fetuses did not affect GSIS, islet size, the proportion of the pancreas comprising ß-cells, or pancreatic or pancreatic islet vascularity. In isolated islet endothelial cells from IUGR fetuses, HGF mRNA expression was not affected by supplemental leucine. CONCLUSIONS: IUGR fetal sheep islets are not responsive to a 9-d leucine infusion with respect to insulin secretion or any histologic features measured. This is in contrast to the response in normally growing fetuses. These results are important when considering nutritional strategies to prevent the adverse islet and ß-cell consequences in IUGR fetuses.
Assuntos
Glucose/farmacologia , Secreção de Insulina/efeitos dos fármacos , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/crescimento & desenvolvimento , Leucina/farmacologia , Animais , Esquema de Medicação , Feminino , Retardo do Crescimento Fetal , Leucina/administração & dosagem , Gravidez , OvinosRESUMO
Pancreatic islet beta cells (ß-cells) synthesize and secrete insulin in response to rising glucose levels and thus are a prime target in both major forms of diabetes. Type 1 diabetes ensues due to autoimmune destruction of ß-cells. On the other hand, the prevailing insulin resistance and hyperglycemia in type 2 diabetes (T2D) elicits a compensatory response from ß-cells that involves increases in ß-cell mass and function. However, the sustained metabolic stress results in ß-cell failure, characterized by severe ß-cell dysfunction and loss of ß-cell mass. Dynamic changes to ß-cell mass also occur during pancreatic development that involves extensive growth and morphogenesis. These orchestrated events are triggered by multiple signaling pathways, including those representing the transforming growth factor ß (TGF-ß) superfamily. TGF-ß pathway ligands play important roles during endocrine pancreas development, ß-cell proliferation, differentiation, and apoptosis. Furthermore, new findings are suggestive of TGF-ß's role in regulation of adult ß-cell mass and function. Collectively, these findings support the therapeutic utility of targeting TGF-ß in diabetes. Summarizing the role of the various TGF-ß pathway ligands in ß-cell development, growth and function in normal physiology, and during diabetes pathogenesis is the topic of this mini-review.
Assuntos
Células Secretoras de Insulina/fisiologia , Ilhotas Pancreáticas/crescimento & desenvolvimento , Fator de Crescimento Transformador beta/fisiologia , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Humanos , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/fisiologia , Organogênese/genética , Transdução de Sinais/fisiologiaRESUMO
Islet vascularization is essential for intact islet function and glucose homeostasis. We have previously shown that primary cilia directly regulate insulin secretion. However, it remains unclear whether they are also implicated in islet vascularization. At eight weeks, murine Bbs4-/-islets show significantly lower intra-islet capillary density with enlarged diameters. Transplanted Bbs4-/- islets exhibit delayed re-vascularization and reduced vascular fenestration after engraftment, partially impairing vascular permeability and glucose delivery to ß-cells. We identified primary cilia on endothelial cells as the underlying cause of this regulation, via the vascular endothelial growth factor-A (VEGF-A)/VEGF receptor 2 (VEGFR2) pathway. In vitro silencing of ciliary genes in endothelial cells disrupts VEGF-A/VEGFR2 internalization and downstream signaling. Consequently, key features of angiogenesis including proliferation and migration are attenuated in human BBS4 silenced endothelial cells. We conclude that endothelial cell primary cilia regulate islet vascularization and vascular barrier function via the VEGF-A/VEGFR2 signaling pathway.
Assuntos
Células Endoteliais/fisiologia , Ilhotas Pancreáticas/irrigação sanguínea , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular , Animais , Linhagem Celular , Feminino , Glucose/metabolismo , Células HEK293 , Humanos , Ilhotas Pancreáticas/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Pancreatic ß-cell dysfunction and reduced insulin secretion play a key role in the pathogenesis of diabetes. Fetal and neonatal islets are functionally immature and have blunted glucose responsiveness and decreased insulin secretion in response to stimuli and are far more proliferative. However, the mechanisms underlying functional immaturity are not well understood. Pancreatic islets are composed of a mixture of different cell types, and the microenvironment of islets and interactions between these cell types are critical for ß-cell development and maturation. RNA sequencing and quantitative proteomic data from intact islets isolated from fetal (embryonic day 19) and 2-week-old Sprague-Dawley rats were integrated to compare their gene and protein expression profiles. Ingenuity Pathway Analysis (IPA) was also applied to elucidate pathways and upstream regulators modulating functional maturation of islets. By integrating transcriptome and proteomic data, 917 differentially expressed genes/proteins were identified with a false discovery rate of less than 0.05. A total of 411 and 506 of them were upregulated and downregulated in the 2-week-old islets, respectively. IPA revealed novel critical pathways associated with functional maturation of islets, such as AMPK (adenosine monophosphate-activated protein kinase) and aryl hydrocarbon receptor signaling, as well as the importance of lipid homeostasis/signaling and neuronal function. Furthermore, we also identified many proteins enriched either in fetal or 2-week-old islets related to extracellular matrix and cell communication, suggesting that these pathways play critical roles in islet maturation. Our present study identified novel pathways for mature islet function in addition to confirming previously reported mechanisms, and provided new mechanistic insights for future research on diabetes prevention and treatment.
Assuntos
Ilhotas Pancreáticas/metabolismo , Proteoma , Transdução de Sinais/fisiologia , Transcriptoma , Animais , Bases de Dados de Proteínas , Perfilação da Expressão Gênica , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/crescimento & desenvolvimento , Proteômica , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: The use of pancreata from pre-weaned piglets has the potential to serve as an unlimited alternative source of islets for clinical xenotransplantation. As pre-weaned porcine islets (PPIs) are immature and require prolonged culture, we developed an islet maturation media (IMM) and evaluated its effect on improving the quantity and quality of PPIs over 14 days of culture. METHODS: PPIs were isolated from the pancreata of pre-weaned Yorkshire piglets (8-15 days old). Each independent islet isolation was divided for culture in either control Ham's F-10 media (n = 5) or IMM (n = 5) for 14 days. On day 3, 7 and 14 of culture, islets were assessed for islet yield, isolation index, viability, insulin content, endocrine cellular composition, differentiation of beta cells, and insulin secretion during glucose stimulation. RESULTS: In comparison to control islets, culturing PPIs in IMM significantly increased islet yield. PPIs cultured in IMM also maintained a stable isolation index and viability throughout 14 days of culture. The insulin content, endocrine cellular composition, and differentiation of beta cells were significantly improved in PPIs cultured in IMM, which subsequently augmented their insulin secretory capacity in response to glucose challenge compared to control islets. CONCLUSIONS: Culturing PPIs in IMM increases islet yield, isolation index, viability, insulin content, endocrine cellular composition, differentiation of endocrine progenitor cells toward beta cells, and insulin secretion. Due to the improved islet quantity and quality after in vitro culture, the use of IMM in the culture of PPIs will assist to advance the outcomes of clinical islet xenotransplantation.
Assuntos
Meios de Cultura , Ilhotas Pancreáticas/crescimento & desenvolvimento , Animais , Animais Recém-Nascidos , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Citometria de Fluxo , Insulina/análise , Insulina/metabolismo , Ilhotas Pancreáticas/química , SuínosRESUMO
Recent reports indicate that 7% of pregnant mothers in North America use cannabis. This is concerning given that in utero exposure to Δ9-tetrahydrocannabinol (Δ9-THC), the main psychoactive component in cannabis, causes fetal growth restriction and may alter replication and survival of pancreatic ß-cells in the offspring. Accordingly, we hypothesized that maternal exposure to Δ9-THC during pregnancy would impair postnatal glucometabolic health of offspring. To test this hypothesis, pregnant Wistar rats were treated with daily intraperitoneal injections of either 3 mg/kg Δ9-THC or vehicle from gestational day 6 to birth. Offspring were subsequently challenged with glucose and insulin at 5 months of age to assess glucose tolerance and peripheral muscle insulin sensitivity. Female offspring exposed to Δ9-THC in utero were glucose intolerant, associated with blunted insulin response in muscle and increased serum insulin concentration 15 min after glucose challenge. Additionally, pancreata from male and female offspring were harvested at postnatal day 21 and 5 months of age for assessment of endocrine pancreas morphometry by immunostaining. This analysis revealed that gestational exposure to Δ9-THC reduced the density of islets in female, but not male, offspring at postnatal day 21 and 5 months, culminating in reduced ß-cell mass at 5 months. These results demonstrate that fetal exposure to Δ9-THC causes female-specific impairments in glucose homeostasis, raising concern regarding the metabolic health of offspring, particularly females, exposed to cannabis in utero.
Assuntos
Dronabinol/toxicidade , Glucose/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal , Animais , Feminino , Homeostase/efeitos dos fármacos , Insulina/metabolismo , Ilhotas Pancreáticas/crescimento & desenvolvimento , Masculino , Troca Materno-Fetal , Gravidez , Ratos Wistar , Caracteres SexuaisRESUMO
It has generally proven challenging to produce functional ß cells in vitro. Here, we describe a previously unidentified protein C receptor positive (Procr+) cell population in adult mouse pancreas through single-cell RNA sequencing (scRNA-seq). The cells reside in islets, do not express differentiation markers, and feature epithelial-to-mesenchymal transition characteristics. By genetic lineage tracing, Procr+ islet cells undergo clonal expansion and generate all four endocrine cell types during adult homeostasis. Sorted Procr+ cells, representing â¼1% of islet cells, can robustly form islet-like organoids when cultured at clonal density. Exponential expansion can be maintained over long periods by serial passaging, while differentiation can be induced at any time point in culture. ß cells dominate in differentiated islet organoids, while α, δ, and PP cells occur at lower frequencies. The organoids are glucose-responsive and insulin-secreting. Upon transplantation in diabetic mice, these organoids reverse disease. These findings demonstrate that the adult mouse pancreatic islet contains a population of Procr+ endocrine progenitors.
Assuntos
Técnicas de Cultura de Células/métodos , Receptor de Proteína C Endotelial/metabolismo , Ilhotas Pancreáticas/citologia , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Feminino , Glucose/metabolismo , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/citologia , Ilhotas Pancreáticas/crescimento & desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Nus , Organoides/crescimento & desenvolvimento , Organoides/metabolismo , Pâncreas/citologia , Pâncreas/metabolismo , Proteína C/metabolismo , Células-Tronco/citologiaRESUMO
Reliance on rodents for understanding pancreatic genetics, development and islet function could limit progress in developing interventions for human diseases such as diabetes mellitus. Similarities of pancreas morphology and function suggest that porcine and human pancreas developmental biology may have useful homologies. However, little is known about pig pancreas development. To fill this knowledge gap, we investigated fetal and neonatal pig pancreas at multiple, crucial developmental stages using modern experimental approaches. Purification of islet ß-, α- and δ-cells followed by transcriptome analysis (RNA-seq) and immunohistology identified cell- and stage-specific regulation, and revealed that pig and human islet cells share characteristic features that are not observed in mice. Morphometric analysis also revealed endocrine cell allocation and architectural similarities between pig and human islets. Our analysis unveiled scores of signaling pathways linked to native islet ß-cell functional maturation, including evidence of fetal α-cell GLP-1 production and signaling to ß-cells. Thus, the findings and resources detailed here show how pig pancreatic islet studies complement other systems for understanding the developmental programs that generate functional islet cells, and that are relevant to human pancreatic diseases.
Assuntos
Diferenciação Celular/genética , Células Secretoras de Insulina/fisiologia , Ilhotas Pancreáticas/embriologia , Ilhotas Pancreáticas/crescimento & desenvolvimento , Suínos , Animais , Animais Recém-Nascidos , Células Cultivadas , Embrião de Mamíferos , Feminino , Feto/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Células Secretoras de Glucagon/citologia , Células Secretoras de Glucagon/fisiologia , Humanos , Ilhotas Pancreáticas/citologia , Camundongos , Organogênese/genética , Gravidez , Suínos/embriologia , Suínos/genética , Suínos/crescimento & desenvolvimento , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TranscriptomaRESUMO
ß-Cell dysfunction and reduction in ß-cell mass are hallmark events of diabetes mellitus. Here we show that ß-cells express abundant Kindlin-2 and deleting its expression causes severe diabetes-like phenotypes without markedly causing peripheral insulin resistance. Kindlin-2, through its C-terminal region, binds to and stabilizes MafA, which activates insulin expression. Kindlin-2 loss impairs insulin secretion in primary human and mouse islets in vitro and in mice by reducing, at least in part, Ca2+ release in ß-cells. Kindlin-2 loss activates GSK-3ß and downregulates ß-catenin, leading to reduced ß-cell proliferation and mass. Kindlin-2 loss reduces the percentage of ß-cells and concomitantly increases that of α-cells during early pancreatic development. Genetic activation of ß-catenin in ß-cells restores the diabetes-like phenotypes induced by Kindlin-2 loss. Finally, the inducible deletion of ß-cell Kindlin-2 causes diabetic phenotypes in adult mice. Collectively, our results establish an important function of Kindlin-2 and provide a potential therapeutic target for diabetes.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Fatores de Transcrição Maf Maior/metabolismo , Proteínas Musculares/metabolismo , beta Catenina/metabolismo , Animais , Proliferação de Células , Proteínas do Citoesqueleto/deficiência , Proteínas do Citoesqueleto/genética , Diabetes Mellitus Experimental/etiologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Insulina/genética , Resistência à Insulina , Ilhotas Pancreáticas/crescimento & desenvolvimento , Ilhotas Pancreáticas/metabolismo , Masculino , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Musculares/deficiência , Proteínas Musculares/genética , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fenótipo , Estabilidade Proteica , beta Catenina/genéticaRESUMO
Existence of stem cells in adult pancreas remains contentious. Single cells suspensions obtained by collagenase and trypsin digestion separately from adult mouse pancreas and pancreatic islets were spun at 1000 rpm (250 g) to collect the cells. At this speed the stem/ progenitor cells remained buoyant and were further enriched by spinning the supernatant at 3000 rpm (1000 g). Two distinct populations of stem cells were detected including pluripotent, very small (2-6 µm) embryonic-like stem cells (VSELs) that expressed nuclear OCT-4A and pluripotent transcripts (Oct-4A, Sox2, Nanog, Stella) and slightly bigger progenitors, pancreatic stem cells (PSCs) that expressed cytoplasmic OCT-4B and PDX-1. Streptozotocin treated diabetic pancreas showed an increase in numbers of VSELs (2-6 µm, 7AAD-, LIN-CD45-SCA1+ cells) and up-regulation of transcripts specific for stem/ progenitor cells. Diabetic mice were further subjected to partial pancreatectomy to study involvement of VSELs/ PSCs during regeneration. VSELs/ PSCs were mobilized in large numbers, were observed in the lumen of blood vessels and PCNA expression suggested their proliferation. Initially, new acini assembled to regenerate the exocrine pancreas and later by Day 30, neogenesis of islets was observed in the vicinity of the blood vessels and pancreatic ducts by the differentiation of endogenous VSELs/ PSCs which may be targeted to regenerate diabetic pancreas in clinical settings.
Assuntos
Diferenciação Celular/genética , Diabetes Mellitus Experimental/terapia , Células-Tronco Embrionárias/transplante , Pâncreas/crescimento & desenvolvimento , Células-Tronco Pluripotentes/transplante , Animais , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/cirurgia , Humanos , Ilhotas Pancreáticas/crescimento & desenvolvimento , Ilhotas Pancreáticas/patologia , Ilhotas Pancreáticas/cirurgia , Camundongos , Pâncreas/patologia , Pâncreas/cirurgia , Pancreatectomia , Regeneração/genética , Transplante de Células-Tronco/métodos , Células-Tronco/metabolismoRESUMO
Statins are cholesterol-lowering agents that increase the incidence of diabetes and impair glucose tolerance via their detrimental effects on nonhepatic tissues, such as pancreatic islets, but the underlying mechanism has not been determined. In atorvastatin (ator)-treated high-fat diet-fed mice, we found reduced pancreatic ß-cell size and ß-cell mass, fewer mature insulin granules, and reduced insulin secretion and glucose tolerance. Transcriptome profiling of primary pancreatic islets showed that ator inhibited the expression of pancreatic transcription factor, mechanistic target of rapamycin (mTOR) signaling, and small G protein (sGP) genes. Supplementation of the mevalonate pathway intermediate geranylgeranyl pyrophosphate (GGPP), which is produced by 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, significantly restored the attenuated mTOR activity, v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) expression, and ß-cell function after ator, lovastatin, rosuvastatin, and fluvastatin treatment; this effect was potentially mediated by sGP prenylation. Rab5a, the sGP in pancreatic islets most affected by ator treatment, was found to positively regulate mTOR signaling and ß-cell function. Rab5a knockdown mimicked the effect of ator treatment on ß-cells. Thus, ator impairs ß-cell function by regulating sGPs, for example, Rab5a, which subsequently attenuates islet mTOR signaling and reduces functional ß-cell mass. GGPP supplementation could constitute a new approach for preventing statin-induced hyperglycemia.
Assuntos
Atorvastatina/farmacologia , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Ácido Mevalônico/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Contagem de Células , Células Cultivadas , Feminino , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/fisiologia , Ilhotas Pancreáticas/crescimento & desenvolvimento , Masculino , Redes e Vias Metabólicas/genética , Camundongos , Camundongos Endogâmicos C57BL , Tamanho do Órgão/genética , Fosfatos de Poli-Isoprenil/farmacologia , Transdução de Sinais/genéticaRESUMO
The pancreas has an essential role in the regulation of glucose homeostasis by secreting insulin, the only hormone with a blood glucose lowering effect in mammals. Several circulating molecules are able to positively or negatively influence insulin secretion. Among them, nutrients such as fatty acids or amino acids can directly act on specific receptors present on pancreatic beta cells. Dietary intake, especially excessive nutrient intake, is known to modify energy balance in adults, resulting in pancreatic dysfunction. However, gestation and lactation are critical periods for fetal development and pup growth and specific dietary nutrients are required for optimal growth. Feeding alterations during these periods will impact offspring development and increase the risk of developing metabolic disorders in adulthood, leading to metabolic programming. This review will focus on the influence of nutrient intake during gestation and lactation periods on pancreas development and function in offspring, highlighting the molecular mechanism of imprinting on this organ.