A radioligand receptor binding assay for measuring of insulin secreted by MIN6 cells after stimulation with glucose, arginine, ornithine, dopamine, and serotonin

Asai, Seiya; Žáková, Lenka; Selicharová, Irena; Marek, Aleš; Jiráček, Jiřı́

doi:10.1007/s00216-021-03423-3

Cited by 13 publications

(12 citation statements)

References 64 publications

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“…In our other recent studies, we analysed the total insulin content in BRIN-BD11, INS-1E and MIN6 cells and in rat Langerhans Islets, and the insulin secretory properties of INS-1E and MIN6 cells after stimulation with glucose and different secretagogues [45]. This revealed that MIN6 cells are more glucose-responsive than INS-1E cells, both in terms of the quantity of secreted insulin and the reproducibility of results, despite a lower total insulin content in these cells.…”

Section: Resultsmentioning

confidence: 99%

Characterization of insulin crystalline form in isolated β-cell secretory granules

et al. 2022

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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 Zn 2+ concentrations inside the pancreatic β-cell insulin secretory granules should promote insulin crystalline state in the form of Zn 2+ -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 4Zn 2+ -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 MIN 6 cell granules in a microcrystalline form, probably consisting of 4Zn 2+ -hexamers of this hormone.

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Section: Resultsmentioning

confidence: 99%

Characterization of insulin crystalline form in isolated β-cell secretory granules

et al. 2022

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“…Binding affinities for IR-B were determined with mouse fibroblasts transfected with human IR-B and with deleted mouse IGF-1R, according to Žáková et al (Zakova, 2014). For both IR-A and IR-B assays, [ 125 I]-monoiodotyrosyl-TyrA14-insulin (2200 Ci/mmol), prepared as described by Asai et al (Asai, 2021), was used as a radiotracer. The dissociation constant of human 125 I-insulin for IR-A and IR-B was 0.3 nM.…”

Section: Methodsmentioning

confidence: 99%

Non-glycosylated IGF2 prohormones are more mitogenic than native IGF2

Potalitsyn

Mrazkova

Selicharová

et al. 2023

Preprint

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Insulin-like Growth Factor-2 (IGF2) is important for the regulation of human embryonic growth and development, and for adults physiology. Incorrect processing of the IGF2 precursor, pro-IGF2(156), leads to the formation of two IGF2 proforms, big-IGF2(87) and big-IGF2(104). Unprocessed and mainly non-glycosylated IGF2 proforms are found at abnormally high levels in certain diseases, but their mode of action is still unclear. Here, we found that pro-IGF2(156) has the lowest ability to form its inactivating complexes with IGF-Binding Proteins and has higher proliferative properties in cells than IGF2 and other IGF prohormones. We also showed that big-IGF2(104) has a seven-fold higher binding affinity for the IGF2 receptor than IGF2, and that pro-IGF2(87) binds and activates specific receptors and stimulates cell growth similarly to the mature IGF2. The properties of these pro-IGF2 forms, especially of pro-IGF2(156) and big-IGF2(104), indicate them as hormones that may be associated with human diseases related to the accumulation of IGF-2 proforms in the circulation.

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“…Binding affinities of analogs for IR‐A and IR‐B were determined by the competition of hormones with [ 125 I]‐monoiodotyrosyl‐TyrA14‐insulin for IR‐A or IR‐B in cell membranes according to Páníková et al 26 Radiolabeled [ 125 I]‐monoiodotyrosyl‐TyrA14‐insulin was prepared according to a procedure described in detail by Asai et al 27 Binding affinities of the analogs for isoform A of the IR (IR‐A) were determined with human IR‐A in human IM‐9 lymphocytes, and binding affinities for IR‐B were determined with mouse fibroblasts transfected with human IR‐B and with deleted mouse IGF‐1R. The individual binding curve for each analog was determined in duplicate points, and the final dissociation constant ( K d ) was calculated from at least three ( n = 3) binding curves (each curve giving a single K d value), determined independently and compared with the binding curves for insulin.…”

Section: Methodsmentioning

confidence: 99%

“…The membranes were probed with anti-phospho-IGF-1Rβ (Tyr1135/1136)/IRβ (Tyr1150/1151) (Cell Signaling Technology) and anti-phospho-Akt (Ser473). Anti-actin (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33) antibody (Sigma-Aldrich, cat.no. A5060) was used as a loading control.…”

Section: Activation Of Ir-a Ir-b and Igf-1r Receptors And Akt Proteinmentioning

confidence: 99%

Targeting the insulin receptor with hormone and peptide dimers

Lin

Selicharová

Mitrová

et al. 2022

Journal of Peptide Science

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Insulin is a key hormone involved in the regulation of overall energetic homeostasis of the organism. The dimeric character of the receptor for insulin evokes ideas about its activation or inhibition with peptide dimers that could either trigger or block the structural transition of the insulin receptor, leading to its activation. Herewith, we present the chemical engineering and biological characterization of several series of insulin dimers or dimers of specific peptides that should be able to bind receptors for insulin or insulin growth factor 1. The hormones or peptides in the dimers were interconnected with different linkers, consisting of triazole moieties and 3, 6, 8, 11, or 23 polyethylene glycol units. The prepared dimers were weaker in binding to insulin receptors than human insulin. However, some of the insulin dimers showed preferential binding specificity toward the isoform A of the insulin receptor, and the insulin dimers also stimulated the insulin receptor more strongly than would be consistent with their binding affinities. Our results suggest that designing insulin dimers may be a promising strategy for modulating the ability of the hormone to activate the receptor or to alter its specificity toward insulin receptor isoforms.

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A radioligand receptor binding assay for measuring of insulin secreted by MIN6 cells after stimulation with glucose, arginine, ornithine, dopamine, and serotonin

Cited by 13 publications

References 64 publications

Characterization of insulin crystalline form in isolated β-cell secretory granules

Characterization of insulin crystalline form in isolated β-cell secretory granules

Non-glycosylated IGF2 prohormones are more mitogenic than native IGF2

Targeting the insulin receptor with hormone and peptide dimers

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