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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

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Abstract

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.

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Funding

The research was supported by the European Regional Development Fund; OP RDE; Project: “Chemical biology for drugging undruggable targets (ChemBioDrug)” (No. CZ.02.1.01/0.0/0.0/16_019/0000729), by Medical Research Council Grant MR/R009066/1. Institutional support was provided by project RVO 61388963 (to the Institute of Organic Chemistry and Biochemistry) of the Czech Academy of Sciences.

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Authors and Affiliations

  1. Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nám. 2, 116 10, Prague 6, Czech Republic

    Seiya Asai, Lenka Žáková, Irena Selicharová, Aleš Marek & Jiří Jiráček

  2. Department of Biochemistry, Faculty of Science, Charles University, 12840, Prague 2, Czech Republic

    Seiya Asai

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  1. Seiya Asai
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  2. Lenka Žáková
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Contributions

Seiya Asai: Investigation and methodology. Lenka Žáková: Supervision and data curation. Irena Selicharová: Validation and data curation. Aleš Marek: Methodology. Jiří Jiráček: Conceptualization, supervision, and writing.

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Correspondence to Jiří Jiráček.

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Source of biological material and animal welfare

All the animal experiments (isolation of pancreatic tissue from Wistar rats) described in this study were performed according to the ethical guidelines for animal experiments and the EU (86/609/EU) and Czech Republic law (Law 246/1992) and were approved by the Committee for experiments with laboratory animals of the Academy of Sciences of the Czech Republic (decision no. 16OZ21899/2020-18134 was issued on December 10, 2020).

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Asai, S., Žáková, L., Selicharová, I. et al. A radioligand receptor binding assay for measuring of insulin secreted by MIN6 cells after stimulation with glucose, arginine, ornithine, dopamine, and serotonin. Anal Bioanal Chem 413, 4531–4543 (2021). https://doi.org/10.1007/s00216-021-03423-3

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