Insulin is stored in vivo inside the pancreatic β-cell insulin secretory granules. In vitro studies have led to an assumption that high insulin and Zn2+ concentrations inside the pancreatic β-cell insulin secretory granules should promote insulin crystalline state in the form of Zn2+-stabilized hexamers. Electron microscopic images of thin sections of the pancreatic β-cells often show a dense, regular pattern core, suggesting the presence of insulin crystals. However, the structural features of the storage forms of insulin in native preparations of secretory granules are unknown, because of their small size, fragile character and difficult handling. We isolated and investigated the secretory granules from MIN6 cells under near-native conditions, using cryo-electron microscopic (Cryo-EM) techniques. The analysis of these data from multiple intra-granular crystals revealed two different rhomboidal crystal lattices. The minor lattice has unit cell parameters (a ≃ b ≃ 84.0 Å, c ≃ 35.2 Å), similar to in vitro crystallized human 4Zn2+-insulin hexamer, whereas the largely prevalent unit cell has more than double c-axis (a ≃ b ≃ c ≃ 96.5 Å) that probably corresponds to two or three insulin hexamers in the asymmetric unit. Our experimental data show that insulin can be present in pancreatic MIN6 cell granules in a microcrystalline form, probably consisting of 4Zn2+-hexamers of this hormone.

• Klíčová slova
• crystallization in vivo, electron microscopy, insulin secretion, peptide hormone, secretory granules, subcellular vesicle,
• MeSH
• beta-buňky * MeSH
• elektronová mikroskopie MeSH
• inzulin MeSH
• Langerhansovy ostrůvky * MeSH
• lidé MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• inzulin MeSH

We adapted a radioligand receptor binding assay for measuring insulin levels in unknown samples. The assay enables rapid and accurate determination of insulin concentrations in experimental samples, such as from insulin-secreting cells. The principle of the method is based on the binding competition of insulin in a measured sample with a radiolabeled insulin for insulin receptor (IR) in IM-9 cells. Both key components, radiolabeled insulin and IM-9 cells, are commercially available. The IR binding assay was used to determine unknown amounts of insulin secreted by MIN6 β cell line after stimulation with glucose, arginine, ornithine, dopamine, and serotonin. The experimental data obtained by the IR binding assay were compared to the results determined by RIA kits and both methods showed a very good agreement of results. We observed the stimulation of glucose-induced insulin secretion from MIN6 cells by arginine, weaker stimulation by ornithine, but inhibitory effects of dopamine. Serotonin effects were either stimulatory or inhibitory, depending on the concentration of serotonin used. The results will require further investigation. The study also clearly revealed advantages of the IR binding assay that allows the measuring of a higher throughput of measured samples, with a broader range of concentrations than in the case of RIA kits. The IR binding assay can provide an alternative to standard RIA and ELISA assays for the determination of insulin levels in experimental samples and can be especially useful in scientific laboratories studying insulin production and secretion by β cells and searching for new modulators of insulin secretion.

• Klíčová slova
• Binding assay, Insulin receptor, Insulin secretion, Radioligand, Secretagogue, β Cells,
• MeSH
• arginin metabolismus   MeSH
• beta-buňky metabolismus   MeSH
• buněčné linie MeSH
• dopamin metabolismus   MeSH
• glukosa metabolismus   MeSH
• inzulin analýza   metabolismus   MeSH
• krysa rodu Rattus MeSH
• Langerhansovy ostrůvky metabolismus   MeSH
• lidé MeSH
• myši MeSH
• ornithin metabolismus   MeSH
• potkani Wistar MeSH
• radioimunoanalýza metody   MeSH
• radioligandová zkouška metody   MeSH
• sekrece inzulinu * MeSH
• serotonin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• arginin MeSH
• dopamin MeSH
• glukosa MeSH
• inzulin MeSH
• ornithin MeSH
• serotonin MeSH

Insulin is produced and stored inside the pancreatic β-cell secretory granules, where it is assumed to form Zn2+-stabilized oligomers. However, the actual storage forms of this hormone and the impact of zinc ions on insulin production in vivo are not known. Our initial X-ray fluorescence experiment on granules from native Langerhans islets and insulinoma-derived INS-1E cells revealed a considerable difference in the zinc content. This led our further investigation to evaluate the impact of the intra-granular Zn2+ levels on the production and storage of insulin in different model β-cells. Here, we systematically compared zinc and insulin contents in the permanent INS-1E and BRIN-BD11 β-cells and in the native rat pancreatic islets by flow cytometry, confocal microscopy, immunoblotting, specific messenger RNA (mRNA) and total insulin analysis. These studies revealed an impaired insulin production in the permanent β-cell lines with the diminished intracellular zinc content. The drop in insulin and Zn2+ levels was paralleled by a lower expression of ZnT8 zinc transporter mRNA and hampered proinsulin processing/folding in both permanent cell lines. To summarize, we showed that the disruption of zinc homeostasis in the model β-cells correlated with their impaired insulin and ZnT8 production. This indicates a need for in-depth fundamental research about the role of zinc in insulin production and storage.

• Klíčová slova
• insulin, pancreatic islets, proinsulin, zinc ions, znt8, β-cells,
• MeSH
• beta-buňky metabolismus   ultrastruktura   MeSH
• chemická frakcionace MeSH
• cytoplazmatická granula metabolismus   MeSH
• exprese genu * MeSH
• glukosa metabolismus   MeSH
• inzulin genetika   metabolismus   MeSH
• krysa rodu Rattus MeSH
• Langerhansovy ostrůvky metabolismus   MeSH
• messenger RNA genetika   metabolismus   MeSH
• průtoková cytometrie metody   MeSH
• zinek metabolismus   MeSH
• zinkový transportér 8 MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• glukosa MeSH
• inzulin MeSH
• messenger RNA MeSH
• Slc30a8 protein, rat MeSH Prohlížeč
• zinek MeSH
• zinkový transportér 8 MeSH