Viral shRNA Knockdown of INS Promotor Activity in EndoC-βH1 Cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE112345 | GEO | 2018/04/06
REPOSITORIES: GEO
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Project description:Viral shRNA Knockdown of INS Promotor Activity in EndoC-βH1 Cells
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Project description:While human enteroviruses are generally regarded as a lytic virus, and persistent non-cytolytic enterovirus infection in pancreatic beta cells has been suspected of playing a role in type 1 diabetes pathogenesis. However, it is still unclear how enteroviruses could exit the pancreatic beta cell in a non-lytic manner. This study aimed to investigate the role of beta cell-derived extracellular vesicles (EVs) in the non-lytic enteroviral spread and infection. Size-exclusion chromatography and antibody-based immunoaffinity purification were used to isolate EVs from echovirus 16-infected human beta EndoC-βH1 cells. EVs were then characterized using transmission electron microscopy and Multiplex Bead-Based Flow Cytometry Assay. Virus production and release were quantified by 50% cell culture infectious dose (CCID50) assay and qRT-PCR. Our results showed that EVs from echovirus 16-infected EndoC-βH1 cells harbor infectious viruses and promote their spread during the pre-lytic phase of infection. Furthermore, the EVs-mediated infection was not inhibited by virus-specific neutralizing antibodies. In summary, this study demonstrated that enteroviruses could exit beta cells non-lytically within infectious EVs, thereby thwarting the access of neutralizing antibodies to viral particles. These data suggest that enterovirus transmission through EVs may contribute to viral dissemination and immune evasion in persistently infected beta cells.
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Project description:4C-seq of insulin promoter, knockdown of INS promoter activity and Genome-wide maps of chromatin state in EndoC-βH1 Cells
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Project description:Insulin (INS) synthesis and secretion from pancreatic β cells are tightly regulated; their deregulation causes diabetes. Here we map INS-associated loci in human pancreatic islets by 4C and 3C techniques and show that the INS gene physically interacts with the SYT8 gene, located over 300 kb away. This interaction is elevated by glucose and accompanied by increases in SYT8 expression. Inactivation of the INS promoter by promoter-targeting siRNA reduces SYT8 gene expression. SYT8-INS interaction and SYT8 transcription are attenuated by CTCF depletion. Furthermore, SYT8 knockdown decreases insulin secretion in islets. These results reveal a non-redundant role for SYT8 in insulin secretion and indicate that the INS promoter acts from a distance to stimulate SYT8 transcription. This suggests a function for the INS promoter in coordinating insulin transcription and secretion through long-range regulation of SYT8 expression in human islets.
2011-02-22 | GSE25862 | GEO
Project description:Investigates the effect of shRNA knock-down of JNK1, JNK2, JNK3 in INS-1 insulin producing cells compared to untransfected INS-1 cells before and after treatment with IL-1beta for 45 min and 6 h.
2014-12-30 | E-MTAB-3146 | biostudies-arrayexpress
Project description:Early 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, 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 data-independent acquisition 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 nonmetastatic insulinomas. EndoC-βH1 secreted 783 proteins in untreated baseline state and 3135 proteins when stressed with nontargeting 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. EndoC-βH1 secretome contained an unexpectedly high proportion of predicted extracellular vesicle proteins. We believe that secretion of extracellular vesicles and bioactive peptides warrant further investigation with specialized proteomics workflows in future studies.
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Project description:Aims/hypothesisStudies on beta cell metabolism are often conducted in rodent beta cell lines due to the lack of stable human beta cell lines. Recently, a human cell line, EndoC-βH1, was generated. Here we investigate stimulus-secretion coupling in this cell line, and compare it with that in the rat beta cell line, INS-1 832/13, and human islets.MethodsCells were exposed to glucose and pyruvate. Insulin secretion and content (radioimmunoassay), gene expression (Gene Chip array), metabolite levels (GC/MS), respiration (Seahorse XF24 Extracellular Flux Analyzer), glucose utilization (radiometric), lactate release (enzymatic colorimetric), ATP levels (enzymatic bioluminescence) and plasma membrane potential and cytoplasmic Ca2+ responses (microfluorometry) were measured. Metabolite levels, respiration and insulin secretion were examined in human islets.ResultsGlucose increased insulin release, glucose utilization, raised ATP production and respiratory rates in both lines, and pyruvate increased insulin secretion and respiration. EndoC-βH1 cells exhibited higher insulin secretion, while plasma membrane depolarization was attenuated, and neither glucose nor pyruvate induced oscillations in intracellular calcium concentration or plasma membrane potential. Metabolite profiling revealed that glycolytic and TCA-cycle intermediate levels increased in response to glucose in both cell lines, but responses were weaker in EndoC-βH1 cells, similar to those observed in human islets. Respiration in EndoC-βH1 cells was more similar to that in human islets than in INS-1 832/13 cells.Conclusions/interpretationFunctions associated with early stimulus-secretion coupling, with the exception of plasma membrane potential and Ca2+ oscillations, were similar in the two cell lines; insulin secretion, respiration and metabolite responses were similar in EndoC-βH1 cells and human islets. While both cell lines are suitable in vitro models, with the caveat of replicating key findings in isolated islets, EndoC-βH1 cells have the advantage of carrying the human genome, allowing studies of human genetic variants, epigenetics and regulatory RNA molecules.
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Project description:Insulin (INS) synthesis and secretion from pancreatic M-NM-2 cells are tightly regulated; their deregulation causes diabetes. Here we map INS-associated loci in human pancreatic islets by 4C and 3C techniques and show that the INS gene physically interacts with the SYT8 gene, located over 300 kb away. This interaction is elevated by glucose and accompanied by increases in SYT8 expression. Inactivation of the INS promoter by promoter-targeting siRNA reduces SYT8 gene expression. SYT8-INS interaction and SYT8 transcription are attenuated by CTCF depletion. Furthermore, SYT8 knockdown decreases insulin secretion in islets. These results reveal a non-redundant role for SYT8 in insulin secretion and indicate that the INS promoter acts from a distance to stimulate SYT8 transcription. This suggests a function for the INS promoter in coordinating insulin transcription and secretion through long-range regulation of SYT8 expression in human islets. Circular Chromosome Conformation Capture (4C)-Seq experiments to profile interactions of INS promoter in human pancreatic islets isolated from two donors: donor 1 and donor 2.
2011-02-22 | E-GEOD-25862 | biostudies-arrayexpress
Project description:An inappropriate diet, particularly excessive consumption of dietary fats and oils, may have a major negative impact on beta-cell function and cause type 2 diabetes mellitus. To investigate this issue, we examined the toxicity of free fatty acid (FFA) compositions mirroring the FFA profiles of various popular edible oils in human EndoC-βH1 beta-cells and in rat islets. For this purpose, we made compositions consisting exclusively of various FFAs in different volumetric percentages mimicking these oils and additionally mixtures of these compositions. Human EndoC-βH1 beta-cells were incubated with different oil compositions and the toxicity, lipid droplet formation, ER-stress, and H2O2 production were analyzed. Compositions with prominent content of saturated as well as unsaturated long-chain FFAs showed moderate but significant toxicity both in human EndoC-βH1 beta-cells and rat islets, however, without further measurable metabolic impairments. On the other hand compositions with high content of medium-chain FFAs revealed no toxicity. A composition with 50% of the very long-chain unsaturated FFA erucic acid caused high toxicity with concomitant peroxisomal H2O2 production. The toxicity of FFAs to human EndoC-βH1 beta-cells was dampened in mixtures of FFA compositions with a significant content of medium-chain FFAs, but not with a significant proportion of unsaturated FFAs.
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Project description:Glucocorticoid use is associated with steroid-induced diabetes mellitus and impaired pancreatic β-cell insulin secretion. Here, the glucocorticoid-mediated transcriptomic changes in human pancreatic islets and the human insulin-secreting EndoC-βH1 cells were investigated to uncover genes involved in β-cell steroid stress-response processes. Bioinformatics analysis revealed glucocorticoids to exert their effects mainly on enhancer genomic regions in collaboration with auxiliary transcription factor families including AP-1, ETS/TEAD, and FOX. Remarkably, we identified the transcription factor ZBTB16 as a highly confident direct glucocorticoid target. Glucocorticoid-mediated induction of ZBTB16 was time- and dose-dependent. Manipulation of ZBTB16 expression in EndoC-βH1 cells combined with dexamethasone treatment demonstrated its protective role against glucocorticoid-induced reduction of insulin secretion and mitochondrial function impairment. In conclusion, we determine the molecular impact of glucocorticoids on human islets and insulin-secreting cells and investigate the effects of glucocorticoid targets on β-cell function. Our findings can pave the way for therapies against steroid-induced diabetes mellitus.
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