Project description:Proteoglycans (PGs) are proteins with glycosaminoglycan (GAG) chains, such as chondroitin sulfate (CS) or heparan sulfate (HS), attached to serine residues. We have earlier shown that prohormones can carry CS, thus, constituting a novel class of PGs. The mapping of GAG modifications of proteins in endocrine cells may thus assist us in delineating possible roles of PGs in endocrine cellular physiology. With this aim, we applied a glycoproteomic approach to identify PGs, their GAG chains and their attachment sites in insulin-secreting cells. Glycopeptides carrying GAG chains were enriched from human pancreatic islets, rat (INS-1 832/13) and mouse (MIN6, NIT-1) insulinoma cell lines by ion exchange chromatography, depolymerized with GAG lyases, and analyzed by nanoflow liquid chromatography-tandem mass spectrometry (nLC-MS/MS). We identified CS modifications of chromogranin-A, islet amyloid polypeptide, secretogranin 1 and secretogranin 2, immunoglobulin superfamily member 10, and protein AMBP. Additionally, we identified two HS-modified prohormones (chromogranin-A and secretogranin-1), which was surprising, as prohormones are not typically regarded as HSPGs. For chromogranin-A, the glycosylation site carried either CS or HS, making it a so-called hybrid site. Additional HS sites were found on syndecan-1, syndecan-4, nerurexin-2, protein NDNF, and testican-1. These results demonstrate that several prohormones, and other constituents of the insulin-secreting cells are PGs. Cell-targeted mapping of the GAG glycoproteome forms an important basis for better understanding of endocrine cellular physiology, and the novel CS and HS sites presented here provide important knowledge for future studies.
Project description:The aim of the present study was to explore the transcriptome of pancreatic islets and, based on this information, to prepare a comprehensive and open access inventory of insulin-producing ?-cell gene expression, the beta-Cell Gene Atlas (BCGA). INS-1 cells, primary rat beta-cells (>87% beta-cells) and non-beta-cells (<3% beta cells, mostly alpha) were isolated by FACS mediated purification of two different rat islet preparations.
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.
Project description:The role of ER Ca2+ release via ryanodine receptors (RyR) in pancreatic B-cell function is not well defined. Deletion of RyR2 from the rat insulinoma INS-1 enhanced the Ca2+ integral (AUC) stimulated by glucose, and reduced levels of the protein IRBIT (AHCYL1). Deletion of IRBIT increased the Ca2+ AUC in response to glucose via enhanced IP3 receptor activity. Insulin content, basal and glucose-stimulated insulin secretion (GSIS), and INS2 mRNA levels were reduced in both RyR2KO and IRBITKO cells. Nuclear localization of S-adenosylhomocysteinase (AHCY) was increased in RyR2KO and IRBITKO cells, and exon 2 of the INS1 and INS2 genes was more extensively methylated in RyR2KO and IRBITKO cells. Deletion of RyR2 or IRBIT resulted in differential regulation of 314 and 137 proteins, respectively, with 41 in common. We conclude that RyR2 regulates IRBIT levels in INS-1 cells, and together regulate insulin content, GSIS, and the proteome, perhaps via DNA methylation.
Project description:There is mounting evidence indicating that piRNAs are also present in somatic cells where they may accomplish additional regulatory tasks. The aim of this study was to identify the piRNAs expressed in pancreatic islets and to determine whether they are involved in the control of beta-cell activities. piRNA profiling of rat pancreatic islets was performed by microarray. We detected about 18’000 piRNAs in rat pancreatic islets, many of which were differentially expressed throughout islet postnatal development.
Project description:To investigate the therapeutic efficacy of beta-cells with IR manipulation, a plasmid containing human IR was stably introduced into rat beta-cell line INS-1 cells (namely, INS-IR cells). In the INS-IR cells, the role of cross-talk between insulin and Wnt signaling pathways in improving the functionality of beta-cells was investigated.
Project description:There is mounting evidence indicating that piRNAs are also present in somatic cells where they may accomplish additional regulatory tasks. The aim of this study was to identify the piRNAs expressed in pancreatic islets and to determine whether they are involved in the control of beta-cell activities. piRNA profiling of rat pancreatic islets was performed by microarray. We detected about 18’000 piRNAs in rat pancreatic islets, many of which were differentially expressed throughout islet of Goto-Kakizaki rats, a well-established model of Type 2 diabetes.
Project description:<p>Application of mass spectrometry enables the detection of metabolic differences between organisms with different nutritional settings. Divergence in the metabolic fingerprints of rat pancreatic INS-1 β-cells were systematically captured with regard to 10 individual essential amino acid (EAA) availability. A high-resolution tandem mass spectrometry system coupled to liquid chromatography produced a horizontal comparison of metabolic profilings of β-cells with individual EAA elevated to 10 mmol/L by turn or removal individual EAA from the medium one by one. Quality control samples were injected at regular intervals throughout the analytical run to monitor and evaluate the stability of the system. The raw data of samples and reference compounds including study protocols have been deposited in the open metabolomics database MetaboLights to enable efficient reuse of the datasets, such as investigating the difference in metabolic process between diverse EAAs as well as screening and verifying potential metabolites affecting insulin secretion and β-cell function.</p>
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.