scholarly journals Characterization of the secretome, transcriptome and proteome of human β cell line EndoC-βH1

2021 ◽  
Author(s):  
Maria Ryaboshapkina ◽  
Kevin Saitoski ◽  
Ghaith M. Hamza ◽  
Andrew F. Jarnuczak ◽  
Claire Berthault ◽  
...  
Keyword(s):  
Cell Line ◽  
Bioactive Peptides ◽  
Cell Model ◽  
Future Research ◽  
List Type ◽  
Diabetes Research ◽  
Β Cells ◽  
Β Cell ◽  
Early Diabetes ◽  
Major Mode

ABSTRACTEarly diabetes research is hampered by limited availability, variable quality and instability of human pancreatic islets in culture. Little is known about the human β cell secretome, and recent studies question translatability of rodent β cell secretory profiles. Here, we verify representativeness of EndoC-βH1, one of the most widely used human β cell lines, as a translational human β cell model based on omics and characterize the EndoC-βH1 secretome. We profiled EndoC-βH1 cells using RNA-seq, Data Independent Acquisition (DIA) and Tandem Mass Tag proteomics of cell lysate. Omics profiles of EndoC-βH1 cells were compared to human β cells and insulinomas. Secretome composition was assessed by DIA proteomics. Agreement between EndoC-βH1 cells and primary adult human β cells was ~90% for global omics profiles as well as for β cell markers, transcription factors and enzymes. Discrepancies in expression were due to elevated proliferation rate of EndoC-βH1 cells compared to adult β cells. Consistently, similarity was slightly higher with benign non-metastatic insulinomas. EndoC-βH1 secreted 671 proteins in untreated baseline state and 3,278 proteins when stressed with non-targeting control siRNA, including known β cell hormones INS, IAPP, and IGF2. Further, EndoC-βH1 secreted proteins known to generate bioactive peptides such as granins and enzymes required for production of bioactive peptides. Unexpectedly, exosomes appeared to be a major mode of secretion in EndoC-βH1 cells. We believe that secretion of exosomes and bioactive peptides warrant further investigation with specialized proteomics workflows in future studies.Graphical abstractHighlightsWe validate EndoC-βH1 as a translational human β cell model using omics.We present the first unbiased proteomics composition of human β cell line secretome.The secretome of human β cells is more extensive than previously thought.Untreated cells secreted 671 proteins and stressed cells secreted 3,278 proteins.Secretion of exosomes and bioactive peptides constitute directions of future research.

scholarly journals Demethylation of the MafB promoter in a compromised β-cell model

2015 ◽  
Vol 55 (1) ◽  
pp. 31-40
Author(s):  
Wataru Nishimura ◽  
Naoko Ishibashi ◽  
Koki Eto ◽  
Nobuaki Funahashi ◽  
Haruhide Udagawa ◽  
...  
Keyword(s):  
Cell Line ◽  
Promoter Activity ◽  
Cell Function ◽  
Cell Model ◽  
Dna Demethylation ◽  
Β Cells ◽  
Β Cell ◽  
Cpg Sites ◽  
Late Passage

Recent studies suggest that dedifferentiation of pancreatic β-cells is involved in compromised β-cell function in diabetes mellitus. We have previously shown that the promoter activity of MafB, which is expressed in α-cells of adult islets and immature β-cells in embryonic pancreas but not in mature β-cells in mice, is increased in compromised β-cells of diabetic model mice. Here, we investigated a rat β-cell line of INS1 cells with late-passage numbers, which showed extremely low expression of MafA and insulin, as an in vitro model of compromised β-cells. In these INS1 cells, the mRNA expression and the promoter activity of MafB were upregulated compared with the early-passage (‘conventional’) INS1 cells. Analysis of the MafB promoter in these late-passage INS1 cells revealed that specific CpG sites in the MafB promoter were partially demethylated. The reporter assay revealed that the unmethylated promoter activity of the 373 bp region containing these CpG sites was higher than the in vitro methylated promoter activity. These results suggest that the chronic culture of the rat β-cell line resulted in partial DNA demethylation of the MafB promoter, which may have a role in MafB promoter activation and possible dedifferentiation in our compromised β-cell model.

scholarly journals NEUROD1 Is Required for the Early α and β Endocrine Differentiation in the Pancreas

2021 ◽  
Vol 22 (13) ◽  
pp. 6713
Author(s):  
Romana Bohuslavova ◽  
Ondrej Smolik ◽  
Jessica Malfatti ◽  
Zuzana Berkova ◽  
Zaneta Novakova ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cell Fate ◽  
Cell Mass ◽  
Neonatal Diabetes ◽  
Diabetes Research ◽  
Pancreas Development ◽  
Β Cells ◽  
Β Cell ◽  
Pancreatic Islet Cell ◽  
Endocrine Differentiation

Diabetes is a metabolic disease that involves the death or dysfunction of the insulin-secreting β cells in the pancreas. Consequently, most diabetes research is aimed at understanding the molecular and cellular bases of pancreatic development, islet formation, β-cell survival, and insulin secretion. Complex interactions of signaling pathways and transcription factor networks regulate the specification, growth, and differentiation of cell types in the developing pancreas. Many of the same regulators continue to modulate gene expression and cell fate of the adult pancreas. The transcription factor NEUROD1 is essential for the maturation of β cells and the expansion of the pancreatic islet cell mass. Mutations of the Neurod1 gene cause diabetes in humans and mice. However, the different aspects of the requirement of NEUROD1 for pancreas development are not fully understood. In this study, we investigated the role of NEUROD1 during the primary and secondary transitions of mouse pancreas development. We determined that the elimination of Neurod1 impairs the expression of key transcription factors for α- and β-cell differentiation, β-cell proliferation, insulin production, and islets of Langerhans formation. These findings demonstrate that the Neurod1 deletion altered the properties of α and β endocrine cells, resulting in severe neonatal diabetes, and thus, NEUROD1 is required for proper activation of the transcriptional network and differentiation of functional α and β cells.

Effects of lipotoxicity on a novel insulin-secreting human pancreatic β-cell line, 1.1B4

2013 ◽  
Vol 394 (7) ◽  
pp. 909-918 ◽  
Author(s):  
Srividya Vasu ◽  
Neville H. McClenaghan ◽  
Jane T. McCluskey ◽  
Peter R. Flatt
Keyword(s):  
Mrna Expression ◽  
Cell Line ◽  
Molecular Mechanisms ◽  
Endoplasmic Reticulum Stress Response ◽  
Future Research ◽  
The Novel ◽  
Pancreatic Β Cell ◽  
Β Cell ◽  
Expression Of Genes ◽  
Cellular Ca

Abstract The novel insulin-secreting human pancreatic β-cell line, 1.1B4, demonstrates stability in culture and many of the secretory functional attributes of human pancreatic β-cells. This study investigated the cellular responses of 1.1B4 cells to lipotoxicity. Chronic 18-h exposure of 1.1B4 cells to 0.5 mm palmitate resulted in decreased cell viability and insulin content. Secretory responses to classical insulinotropic agents and cellular Ca2+ handling were also impaired. Palmitate decreased glucokinase activity and mRNA expression of genes involved in secretory function but up-regulated mRNA expression of HSPA5, EIF2A, and EIF2AK3, implicating activation of the endoplasmic reticulum stress response. Palmitate also induced DNA damage and apoptosis of 1.1B4 cells. These responses were accompanied by increased gene expression of the antioxidant enzymes SOD1, SOD2, CAT and GPX1. This study details molecular mechanisms underlying lipotoxicity in 1.1B4 cells and indicates the potential value of the novel β-cell line for future research.

scholarly journals Differentiation of Sendai Virus-Reprogrammed iPSC into β Cells, Compared with Human Pancreatic Islets and Immortalized β Cell Line

2018 ◽  
Vol 27 (10) ◽  
pp. 1548-1560 ◽  
Author(s):  
Silvia Pellegrini ◽  
Fabio Manenti ◽  
Raniero Chimienti ◽  
Rita Nano ◽  
Linda Ottoboni ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pancreatic Islets ◽  
Cell Line ◽  
Expression Profile ◽  
Sendai Virus ◽  
Secretory Function ◽  
Β Cells ◽  
Β Cell ◽  
Insulin Producing Cells ◽  
Gene And Protein Expression

Background: New sources of insulin-secreting cells are strongly in demand for treatment of diabetes. Induced pluripotent stem cells (iPSCs) have the potential to generate insulin-producing cells (iβ). However, the gene expression profile and secretory function of iβ still need to be validated in comparison with native β cells. Methods: Two clones of human iPSCs, reprogrammed from adult fibroblasts through integration-free Sendai virus, were differentiated into iβ and compared with donor pancreatic islets and EndoC-βH1, an immortalized human β cell line. Results: Both clones of iPSCs differentiated into insulin+ cells with high efficiency (up to 20%). iβ were negative for pluripotency markers (Oct4, Sox2, Ssea4) and positive for Pdx1, Nkx6.1, Chromogranin A, PC1/3, insulin, glucagon and somatostatin. iβ basally secreted C-peptide, glucagon and ghrelin and released insulin in response either to increasing concentration of glucose or a depolarizing stimulus. The comparison revealed that iβ are remarkably similar to donor derived islets in terms of gene and protein expression profile and similar level of heterogeneity. The ability of iβ to respond to glucose instead was more related to that of EndoC-βH1. Discussion: We demonstrated that insulin-producing cells generated from iPSCs recapitulate fundamental gene expression profiles and secretory function of native human β cells.

GENETICALLY ENGINEERED PIG MODELS FOR TRANSLATIONAL DIABETES RESEARCH

2013 ◽  
Vol 25 (1) ◽  
pp. 320 ◽  
Author(s):  
Eckhard Wolf
Keyword(s):  
Mouse Models ◽  
Cell Mass ◽  
Physiological Age ◽  
Diabetic Patients ◽  
Diabetes Research ◽  
Rodent Models ◽  
Β Cells ◽  
Β Cell ◽  
Transgenic Pigs ◽  
Genetic Modifications

Animal models play crucial roles for understanding disease mechanisms and for the development and evaluation of therapeutic strategies. In biomedicine, classical rodent models are most widely used for several reasons, including standardization of genetics and environment, cost efficiency, and the possibility to introduce targeted genetic modifications for the generation of tailored disease models. However, due to differences in anatomical and physiological characteristics, rodent models do not always reflect the situation of human patients sufficiently well to be predictive in terms of efficacy and safety of new therapies. In this respect, the pig has been discussed as a missing link between mouse models and human patients. As a monogastric omnivore, the pig shares many anatomical and physiological similarities with humans. Importantly, the techniques for genetic modification of pigs have been refined to a level allowing almost the same spectrum of alterations as in mouse models (Aigner et al. 2010 J. Mol. Med. (Berl.) 88, 653–664). These include inducible transgene expression systems (Klymiuk et al. 2012 FASEB J. 26, 1086–1099) as well as the introduction of targeted genetic modifications (Klymiuk et al. 2012 J. Mol. Med. (Berl.) 90, 597–608). A major focus of our laboratory is the generation, characterisation, and implementation of pig models for translational diabetes research. Transgenic pigs expressing a dominant negative receptor for the incretin hormone glucose-dependent insulinotropic polypeptide (GIP) demonstrated a crucial role of the GIP system for the physiological age-related expansion of pancreatic β-cell mass. Moreover, this animal model shares important characteristics of type 2 diabetes mellitus: impaired incretin effect, reduced glucose tolerance and insulin secretion, and a progressive reduction of β-cell mass (Renner et al. 2010 Diabetes 59, 1228–1238). More recently, we used this model to search for metabolic biomarkers which are associated with progression in the pre-diabetic period and identified specific amino acid and lipid signatures as candidate biomarkers (Renner et al. 2012 Diabetes 61, 2166–2175). Further, we created the first pig model for permanent neonatal diabetes by expression C94Y mutant insulin in the β-cells of transgenic pigs. In addition to their use as biomedical models, pigs may also serve as organ and tissue donors for xenotransplantation. Transplantation of encapsulated porcine pancreatic islets to type 1 diabetic patients with severe unaware hypoglycemia has already entered clinical studies, but encapsulation may shorten the lifespan of the islets. Therefore, in order to overcome the rejection of pig islets by human T-cells, we generated transgenic pigs expressing the optimized CTLA-4Ig variant LEA29Y in the pancreatic β-cells. Islets from LEA29Y transgenic pigs rescued diabetes and were protected against rejection in a humanized mouse model (Klymiuk et al. 2012 Diabetes 61, 1527–1532).

scholarly journals MECHANISMS IN ENDOCRINOLOGY: Alternative splicing: the new frontier in diabetes research

2016 ◽  
Vol 174 (5) ◽  
pp. R225-R238 ◽  
Author(s):  
Jonàs Juan-Mateu ◽  
Olatz Villate ◽  
Décio L Eizirik
Keyword(s):  
Alternative Splicing ◽  
Cell Fate ◽  
Immune Cells ◽  
Regulatory Networks ◽  
Protein Isoforms ◽  
Diabetes Research ◽  
Β Cells ◽  
Β Cell ◽  
Pancreatic Β Cells

Type 1 diabetes (T1D) is a chronic autoimmune disease in which pancreatic β cells are killed by infiltrating immune cells and by cytokines released by these cells. This takes place in the context of a dysregulated dialogue between invading immune cells and target β cells, but the intracellular signals that decide β cell fate remain to be clarified. Alternative splicing (AS) is a complex post-transcriptional regulatory mechanism affecting gene expression. It regulates the inclusion/exclusion of exons into mature mRNAs, allowing individual genes to produce multiple protein isoforms that expand the proteome diversity. Functionally related transcript populations are co-ordinately spliced by master splicing factors, defining regulatory networks that allow cells to rapidly adapt their transcriptome in response to intra and extracellular cues. There is a growing interest in the role of AS in autoimmune diseases, but little is known regarding its role in T1D. In this review, we discuss recent findings suggesting that splicing events occurring in both immune and pancreatic β cells contribute to the pathogenesis of T1D. Splicing switches in T cells and in lymph node stromal cells are involved in the modulation of the immune response against β cells, while β cells exposed to pro-inflammatory cytokines activate complex splicing networks that modulate β cell viability, expression of neoantigens and susceptibility to immune-induced stress. Unveiling the role of AS in β cell functional loss and death will increase our understanding of T1D pathogenesis and may open new avenues for disease prevention and therapy.

scholarly journals Photolysis of cell-permeant caged inositol pyrophosphates controls oscillations of cytosolic calcium in a β-cell line

2019 ◽  
Vol 10 (9) ◽  
pp. 2687-2692 ◽  
Author(s):  
S. Hauke ◽  
A. K. Dutta ◽  
V. B. Eisenbeis ◽  
D. Bezold ◽  
T. Bittner ◽  
...  
Keyword(s):  
Cell Line ◽  
Cytosolic Calcium ◽  
Β Cells ◽  
Β Cell ◽  
Inositol Pyrophosphate ◽  
Inositol Pyrophosphates ◽  
Line P

β-Cells respond directly to the intracellular photochemical release of caged inositol pyrophosphate isomers with modulations of oscillations in cytosolic Ca2+.

scholarly journals Insulin and secretagogues differentially regulate fluid-phase pinocytosis in insulin-secreting β-cells

1996 ◽  
Vol 318 (2) ◽  
pp. 623-629 ◽  
Author(s):  
Gang XU ◽  
Jennie HOWLAND ◽  
Paul L ROTHENBERG
Keyword(s):  
T Cells ◽  
Cell Surface ◽  
Insulin Receptor ◽  
Cell Line ◽  
Chinese Hamster Ovary Cells ◽  
Insulin Receptors ◽  
Fluid Phase ◽  
Β Cells ◽  
Β Cell ◽  
Basal Rate

The physiological role of the β-cell insulin receptor is unknown. To evaluate a candidate function, the insulin regulation of fluid-phase pinocytosis was investigated in a clonal insulinoma cell line (βTC6-F7) and, for comparison, also in Chinese hamster ovary cells transfected with the human insulin receptor (CHO-T cells). In CHO-T cells, the net rate of fluid-phase pinocytosis was rapidly increased 3–4-fold over the basal rate by 100 nM insulin, with half-maximal stimulation at 2 nM insulin, as assayed by cellular uptake of horseradish peroxidase from the medium. Wortmannin, an inhibitor of phosphatidylinositol (PI)-3-kinase, blocked insulin-stimulated pinocytosis with an IC50 of 7.5 nM without affecting the basal rate of pinocytosis. In insulin-secreting βTC6-F7 cells, the secretagogues glucose and carbachol (at maximally effective concentrations of 15 mM and 0.5 mM respectively) augmented fluid-phase pinocytosis 1.65-fold over the basal rate. Wortmannin also inhibited secretagogue-stimulated pinocytosis in these β-cells with an IC50 of 7 nM but did not affect the basal rate of pinocytosis measured in the absence of secretagogues. Wortmannin did not influence either basal or secretagogue-induced insulin secretion. Although these βTC6-F7 cells have cell-surface insulin receptors, adding exogenous insulin or insulin-like growth factor 1 did not affect their rate of fluid-phase pinocytosis, either in the absence or presence of secretagogues. From these observations, we conclude that: (1) in both insulin-secreting β-cells and in conventional, insulin-responsive CHO-T cells, a common, wortmannin-sensitive reaction, which probably involves PI-3-kinase, regulates fluid-phase pinocytosis; (2) the insulin-receptor signal transduction pathway is dissociated from the regulation of fluid-phase pinocytosis in the insulin-secreting β-cell line we studied; and (3) the enhancement of fluid-phase pinocytosis associated with secretagogue-induced insulin release in βTC6-F7 cells is not attributable to autocrine activation of β-cell surface insulin receptors.

scholarly journals Dual-Reporter β-Cell-Specific Male Transgenic Rats for the Analysis of β-Cell Functional Mass and Enrichment by Flow Cytometry

Endocrinology ◽  
2015 ◽  
Vol 157 (3) ◽  
pp. 1299-1306 ◽  
Author(s):  
Julien Ghislain ◽  
Ghislaine Fontés ◽  
Caroline Tremblay ◽  
Melkam A. Kebede ◽  
Vincent Poitout
Keyword(s):  
Insulin Secretion ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Yellow Fluorescent Protein ◽  
Cell Mass ◽  
Renilla Luciferase ◽  
Diabetes Research ◽  
Β Cells ◽  
Transgenic Rats ◽  
Β Cell

Abstract Mouse β-cell-specific reporter lines have played a key role in diabetes research. Although the rat provides several advantages, its use has lagged behind the mouse due to the relative paucity of genetic models. In this report we describe the generation and characterization of transgenic rats expressing a Renilla luciferase (RLuc)-enhanced yellow fluorescent protein (YFP) fusion under control of a 9-kb genomic fragment from the rat ins2 gene (RIP7-RLuc-YFP). Analysis of RLuc luminescence and YFP fluorescence revealed that reporter expression is restricted to β-cells in the adult rat. Physiological characteristics including body weight, fat and lean mass, fasting and fed glucose levels, glucose and insulin tolerance, and β-cell mass were similar between two RIP7-RLuc-YFP lines and wild-type littermates. Glucose-induced insulin secretion in isolated islets was indistinguishable from controls in one of the lines, whereas surprisingly, insulin secretion was defective in the second line. Consequently, subsequent studies were limited to the former line. We asked whether transgene activity was responsive to glucose as shown previously for the ins2 gene. Exposing islets ex vivo to high glucose (16.7 mM) or in vivo infusion of glucose for 24 hours increased luciferase activity in islets, whereas the fraction of YFP-positive β-cells after glucose infusion was unchanged. Finally, we showed that fluorescence-activated cell sorting of YFP-positive islet cells can be used to enrich for β-cells. Overall, this transgenic line will enable for the first time the application of both fluorescence and bioluminescence/luminescence-based approaches for the study of rat β-cells.

scholarly journals CRISPR/Cas9-Mediated α-ENaC Knockout in a Murine Pancreatic β-Cell Line

2021 ◽  
Vol 12 ◽  
Author(s):  
Xue Zhang ◽  
Lihua Zhao ◽  
Runbing Jin ◽  
Min Li ◽  
Mei-Shuang Li ◽  
...  
Keyword(s):  
Cell Line ◽  
Gene Knockout ◽  
Pancreatic Tissue ◽  
Pancreatic Β Cell ◽  
Β Cells ◽  
Β Cell ◽  
Pancreatic Β Cells ◽  
Min6 Cells ◽  
Insulin Synthesis ◽  
Guide Rna

Many ion channels participate in controlling insulin synthesis and secretion of pancreatic β-cells. Epithelial sodium channel (ENaC) expressed in human pancreatic tissue, but the biological role of ENaC in pancreatic β-cells is still unclear. Here, we applied the CRISPR/Cas9 gene editing technique to knockout α-ENaC gene in a murine pancreatic β-cell line (MIN6 cell). Four single-guide RNA (sgRNA) sites were designed for the exons of α-ENaC. The sgRNA1 and sgRNA3 with the higher activity were constructed and co-transfected into MIN6 cells. Through processing a series of experiment flow included drug screening, cloning, and sequencing, the α-ENaC gene-knockout (α-ENaC−/−) in MIN6 cells were obtained. Compared with the wild-type MIN6 cells, the cell viability and insulin content were significantly increased in α-ENaC−/− MIN6 cells. Therefore, α-ENaC−/− MIN6 cells generated by CRISPR/Cas9 technology added an effective tool to study the biological function of α-ENaC in pancreatic β-cells.

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