Issue 12, 2019

Insulin sensor cells for the analysis of insulin secretion responses in single living pancreatic β cells

Abstract

Investigation of the functions of insulin-secreting cells in response to glucose in single-living cells is essential for improving our knowledge on the pathogenesis of diabetes. Therefore, it is desired to develop a new convenient method that enables the direct detection of insulin secreted from single-living cells. Here, insulin-sensor-cells expressing a protein-based insulin-detecting probe immobilized on the extracellular membrane were developed to evaluate the insulin-secretion response in single-living pancreatic β cells. The protein-based insulin-detecting probe (NαLY) was composed of a bioluminescent protein (nano-luc), the αCT segment of the insulin receptor, L1 and CR domains of the insulin receptor, and a fluorescent protein (YPet). NαLY exhibited a bioluminescence resonance energy transfer (BRET) signal in response to insulin; thus, cells of Hepa1–6 line were genetically engineered to express NαLY on the extracellular membrane. The cells were found to act as insulin-sensor-cells, exhibiting a BRET signal in response to insulin. When the insulin-sensor-cells and pancreatic β cells (MIN6 cell line) were cocultured and stimulated with glucose, insulin-sensor-cells nearby pancreatic β cells showed the spike-shaped BRET signal response, whereas the insulin-sensor-cells close to one pancreatic β cell did not exhibit such signal response. However, all the insulin-sensor-cells showed a gradual increase in BRET signals, which were presumably attributed to the increase in insulin concentrations in the culture dish, confirming the function of these insulin-sensor-cells. Therefore, we demonstrated that heterogenetic insulin secretion in single-living pancreatic β cells could be measured directly using the insulin sensor cells.

Graphical abstract: Insulin sensor cells for the analysis of insulin secretion responses in single living pancreatic β cells

Supplementary files

Article information

Article type
Paper
Submitted
02 Cig 2019
Accepted
08 Cax 2019
First published
09 Cax 2019

Analyst, 2019,144, 3765-3772

Insulin sensor cells for the analysis of insulin secretion responses in single living pancreatic β cells

H. Shigeto, T. Ono, T. Ikeda, R. Hirota, T. Ishida, A. Kuroda and H. Funabashi, Analyst, 2019, 144, 3765 DOI: 10.1039/C9AN00405J

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